Deorphanization of mouse bitter taste receptors · 5/20/2016 · bitter taste receptor genes. It is assumed that the orthologous bitter taste receptor genes mediate the recognition

Deorphanization of mouse bitter taste receptors

1

Comprehensive analysis of mouse bitter taste receptors reveals different molecular receptive ranges for

orthologous receptors in mice and humans

Kristina Lossow1, Sandra Hübner1, Natacha Roudnitzky1, Jay P. Slack2, Federica Pollastro3, Maik Behrens1, and Wolfgang Meyerhof1

1Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee

114-116, 14558 Nuthetal, Germany 2Givaudan Flavors Corporation, 1199 Edison Drive, Cincinnati, 45216 Ohio, USA

3Department of Drug Sciences, University of Eastern Piemonte, 28100 Novara, Italy

Running title: Deorphanization of mouse bitter taste receptors To whom correspondence should be addressed: Maik Behrens, Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany. Telephone: (+49) 33200 88 2545; FAX: (+49) 33200 88 2384; E-mail: [email protected] Keywords: bitter taste receptors, G protein-coupled receptors, heterologous expression, calcium imaging, mouse, human

http://www.jbc.org/cgi/doi/10.1074/jbc.M116.718544The latest version is at JBC Papers in Press. Published on May 20, 2016 as Manuscript M116.718544

Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on Novem

ber 29, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/cgi/doi/10.1074/jbc.M116.718544

http://www.jbc.org/


2

Abstract One key to animal survival is the detection and avoidance of potentially harmful compounds by their bitter taste. Variable numbers of taste 2 receptor genes expressed in the gustatory end organs enable bony vertebrates (Euteleostomi) to recognize numerous bitter chemicals. It is believed that the receptive ranges of bitter taste receptor repertoires match the profiles of bitter chemicals that the species encounter in their diets. Human and mouse genomes contain pairs of orthologous bitter receptor genes that have been conserved throughout evolution. Moreover, expansions in both lineages generated species-specific sets of bitter taste receptor genes. It is assumed that the orthologous bitter taste receptor genes mediate the recognition of bitter toxins relevant for both species, whereas the lineage-specific receptors enable the detection of substances differently encountered by mice and humans. By challenging 34 mouse bitter taste receptors with 128 prototypical bitter substances in a heterologous expression system we identified cognate compounds for 21 receptors, 19 of which were previously orphan receptors. We demonstrate that mouse taste 2 receptors, like their human counterparts, vary greatly in their breadth of tuning, ranging from very broadly to extremely narrowly tuned receptors. However, when compared to humans, mice possess fewer broadly tuned receptors and an elevated number of narrowly tuned receptors, supporting the idea that a large receptor repertoire is the basis for the evolution of specialized receptors. Moreover, we demonstrate that sequence-orthologous bitter taste receptors have distinct agonist profiles. Species-specific gene expansions have enabled further diversification of bitter substance recognition spectra. Introduction The plethora of natural compounds that taste bitter for humans comprises numerous chemicals with pharmacological activities that can make them powerful toxins such as the alkaloids strychnine and colchicine, or the sesquiterpene lactone picrotoxinin (1). However, compounds believed to exert health beneficial effects such as the antioxidative phytoestrogen genistein from soy (2), the analgesic drug acetaminophen (3) or various polyphenols also taste bitter (4). To avoid

ingestion of bitter substances which would pose a threat to organisms, efficient recognition and rejection mechanisms have developed throughout the animal kingdom. In bony vertebrates (Euteleostomi), the avoidance of bitter compounds is centered on taste receptors that detect potentially harmful substances with high accuracy and adequate sensitivity (5). Vertebrate bitter taste receptors, called taste 2 receptors (Tas2r), are G protein-coupled receptors (GPCR) only remotely related to other classes of this large and enormously versatile receptor family (6-10). During evolution the first Tas2r genes appeared in the genomes of bony fish (11). In higher vertebrates frequent independent expansions and pseudogenization events resulted in differently sized Tas2r gene repertoires (12). Consequently, the number of putatively functional Tas2r genes varies considerably in vertebrates, ranging from 0 in baleen and tooth whales as well as penguins (13-16) to more than 50 in Western clawed frogs and 80 in Coelacanth (17-20). Thus, humans with ~25 and mice with ~35 putatively functional members possess averagely sized Tas2r repertoires (21). The human genome not only contains fewer intact TAS2R genes than the mouse genome, but also a larger number of pseudogenes (11 in human vs. 7 in mice). This has been interpreted as a sign for relaxed selective constraints on the human TAS2R gene repertoire (22). The Tas2r genes of human and mouse occur clustered at few syntenic chromosomal regions (6,22,23). The majority of Tas2r genes located on human chromosome 12 and mouse chromosome 6, respectively, occur in clusters of species-specific bitter taste receptor genes, which likely arose from gene duplications after the divergence of primate and rodent lineages. It has been speculated that these lineage-specific Tas2rs recognize toxic bitter substances of particular relevance for the corresponding species (23). In contrast, the majority of Tas2r genes located on human chromosomes 5 and 7 and mouse chromosomes 2 and 15, respectively, exhibit a one-to-one orthology suggesting that they developed prior to the divergence of primate and rodent lineages and enable the recognition of bitter substances equally important to humans and mice (23). If the above hypothesis is true, human and mouse should share Tas2rs with conserved agonists, namely the one-to-one orthologs, and possess others with cognate

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


3

bitter substances mostly relevant to one of the two species. In fact, when interpreting data of rodent behavioral experiments, it is frequently argued that the murine Tas2rs with highest sequence identity are true functional orthologs of their human counterparts recognizing the same bitter compounds (cf. (24-28)). However, structure function analyses of human bitter taste receptors revealed that very few differences in the amino acid sequences of TAS2Rs can account for largely deviating agonist spectra (29). Conversely, human TAS2R paralogs with pronounced amino acid sequence differences can have agonists in common even though they recognize these compounds by different binding modes (30). The ~25 human bitter taste receptors (TAS2Rs) are comparatively well characterized with identified agonists for 21 of the ~25 receptors (1-4,7,30-42). Collectively, these data indicated that humans have 3 very broadly tuned TAS2R ‘generalists’ and 8 receptor ‘specialists’ that are narrowly tuned. Moreover, they have two TAS2Rs for compounds sharing structural motifs as well as 8 moderately tuned receptors. Recently, the bitter taste receptor gene repertoires of chicken, turkey, zebra finch, and the Western clawed frog have been functionally analyzed. These studies revealed that narrowly tuned Tas2rs were only found in species with larger Tas2r gene numbers such as frog and zebra finch, whereas the 3 chicken and 2 turkey receptors were all broadly tuned (17). In mice, agonists have been reported only for 2 of the 35 putatively functional Tas2rs, leaving the receptive range of the mouse Tas2r repertoire uncharacterized. For Tas2r105 an inhibitor of mRNA translation, cycloheximide, has been identified as a specific and potent agonist (7). The other receptor is Tas2r108, which is activated by denatonium benzoate and 6-n-propyl-2-thiouracil (PROP) with low potency (7). Thus, the scarcity of data on the functional properties of mouse Tas2rs does not provide clear insight into the extent of functional orthology or whether species-specific Tas2r gene expansions have indeed resulted in specialized Tas2rs for bitter compounds of species-specific relevance. In order to close this gap in knowledge we investigated whether the putatively functional murine Tas2rs respond to an array of 128 bitter substances in a functional heterologous expression assay. Behavioral experiments were also

performed to correlate the tuning properties of mouse Tas2rs with avoidance behavior of the animals assessed by brief-access taste tests. Results All Tas2rs genes are expressed in the posterior papillae of the mouse tongue To elucidate if all mouse Tas2rs are indeed expressed in gustatory cells of the tongue and to determine their relative expression levels and patterns, we performed quantitative RT-PCR (qRT-PCR) and in situ hybridization experiments. Quantitative RT-PCR expression analyses demonstrated that all Tas2r genes are expressed in the epithelium of the posterior tongue (Figure 1A). Whereas some receptor mRNAs, e.g. those for Tas2r108, Tas2r118, Tas2r126, Tas2r135, and Tas2r137, were quite abundant reaching ~20% of the α-gustducin mRNA level, other Tas2r mRNAs such as those of Tas2r114, Tas2r122, and Tas2r140, were rare, just reaching detection levels. The majority of Tas2r genes exhibited intermediate expression levels. Differences in Tas2r gene expression for six selected receptor mRNAs were also evident on a cellular level as shown by in situ hybridization experiments with mouse vallate papillae sections (Figure 1B). For example, Tas2r118 mRNA was detected in a large subset of vallate taste cells with rather strong signal intensity, while Tas2r105 stained fewer cells but with comparable signal intensity. The Tas2r138 and Tas2r115 probes labeled cells that resemble Tas2r105 positive cells in number but not in staining intensity. Finally, cells expressing Tas2r120 or Tas2r102 were rare and showed faint staining. Alpha-gustducin mRNA was detected in many cells with strong signal intensity as anticipated for a gene coding for a common signaling component of sweet, umami, and bitter taste transduction (9,43-45). Vallate papillae sections treated with sense probes showed no staining confirming specificity of the detection method. The results obtained by qRT-PCR and by in situ hybridization show a good correlation. The receptor Tas2r118 showing the strongest expression in the qRT-PCR experiment also exhibited the most pronounced staining in the in situ hybridization experiment. This is also true for Tas2r105 and Tas2r138 which exhibit lower mRNA levels detected by qRT-PCR and fewer

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


4

cells stained in the in situ hybridization experiments. For the two receptors, Tas2r115 and Tas2r120, which showed low mRNA levels in the qRT-PCR experiment, also only faint signals were obtained by in situ hybridization. Only Tas2r102 which, in the qRT-PCR experiment, exhibited mRNA levels comparable to that of Tas2r105 demonstrated lower in situ hybridization signal intensity. Tuning breadth of mouse Tas2rs For many decades aversive behavior has been studied in mice as model organisms to investigate taste responses elicited by compounds that taste bitter to humans (46-54). However, unlike human psychophysical observations which can be correlated with data from the functional characterization of human TAS2Rs (e.g. (3)), the lack of data on agonist specificities of mouse Tas2rs prevented similar correlations in mouse with few exceptions (7). To identify cognate agonists for mouse Tas2rs, we functionally expressed 34 of the 35 putatively functional mouse Tas2rs (excluding Tas2r116) in heterologous cells and screened them for responsiveness to a set of 128 predominantly naturally occurring bitter compounds with diverse chemical structures (Supplementary table 2S). The majority of the tested substances were selected from a bitter compound library used recently for the screening of human TAS2Rs (3,42), and subsequently for Tas2rs from avian and frog species (17). These experiments allowed us to identify cognate agonists for 21 of the 34 examined receptors (Table 1, Figure 2). Tas2r105 appears to be a ‘generalist’ receptor responding to a broad panel of 45 substances (35%) of the library (Table 1, Figure 3, for selected concentration response curves). Three other mouse bitter taste receptors, Tas2r121 (11%), Tas2r135 (9%), and Tas2r144 (16%), were also responsive to a large number of different bitter compounds (Table 1). Further 10 Tas2rs recognized between 4 and 10 substances, seven receptors turned out to be ‘specialists’ with strongly restricted agonist profiles responding to only 1 to 3 test compounds, whereas 13 receptors were not activated by any of the test substances. We then challenged these 13 Tas2rs with additional 77 agonists (shown in italic, Supplementary table 2S) in order to increase the

probability to deorphan them but did not register any further responses. To figure out, if failure to deorphan might be due to insufficient cell surface expression, a selected subset of Tas2rs was subjected to immunocytochemistry. The majority of the selected receptors show a clear external localization of Rho-epitopes added to the N-terminus of the recombinant Tas2r when unpermeabilized cells were subjected to staining (Figure 6, Table 3). Other receptors, Tas2r102 and Tas2r131, were only visible after permeabilization. Since we did not identify an agonist for these receptors, insufficient cell surface expression may indeed have prevented deorphanization. However, the receptors Tas2r106, Tas2r118, and Tas2r134, for which we also did not identify an agonists, exhibited clear cell surface staining of unpermeabilized cells (Figure 6, Table 3), indicating that problems in receptor routing are not the dominant reason for preventing functional identification of agonists. We have not found a Tas2r that lacks cell surface staining but resulted in the identification of agonists. Tas2r specific bitter compounds Despite their partially overlapping agonist profiles, each of the mouse Tas2rs was activated by a unique subset of test substances. Moreover, regardless of their tuning breadth, 8 Tas2rs have specific cognate agonists not detected by any of the other identified 13 Tas2rs (Table 1, underlined). For example, 16 out of the 45 Tas2r105 activators were specific including cycloheximide, which is in full agreement with the crucial role of Tas2r105 in cycloheximide avoidance (7). Interestingly, also 4 structurally related N-acyl homoserine lactones (AHLs) exclusively activated Tas2r105. These compounds are crucially involved in bacterial quorum sensing and induce antibacterial responses of the host (55). Concentration ranges for activators of mouse Tas2rs Two parameters are decisive in order to compare receptors and to predict the physiological importance of a receptor. The potency is a measure of the concentration range at which the receptor becomes responsive, whereas efficacy refers to the strength of the induced receptor’s

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


5

response. Our data demonstrate that different agonists activated the mouse Tas2rs with widely different efficacies and potencies, which is illustrated by the different maximal signal amplitudes of the activated receptors (Supplementary table 3S/A) as well as by their threshold concentrations (Table 1) and EC50-values (Supplementary table 3S/B). Several cognate Tas2rs for the majority of bitter compounds We also observed that most bitter compounds activate several mouse Tas2rs. The number of Tas2rs sensitive to an individual bitter compound varied considerably (Table 1). The substances that activated the highest number of Tas2rs were quinine and sucralose with 7 receptors, followed by the two synthetic bitter compounds PROP and diphenidol that elicited responses of 6 receptors, respectively. Whereas 5 substances, among them denatonium benzoate (Figure 4) activated 5 receptors. Hence, the ability of different bitter compounds to stimulate several mouse Tas2rs is similar to the response pattern seen for human TAS2Rs (3), although overall promiscuity was greater in human TAS2Rs. Screening of human receptors with newly identified mouse Tas2r agonists Since we analyzed more bitter compounds in this study than in our previous characterization of the human TAS2Rs (3,42), we performed an additional screening experiment with the 25 putatively functional human TAS2Rs and those substances not included in our previous analyses (Supplementary table 3S/C) in order to broaden the basis for comparisons. While this screening failed to identify activators for any of the 4 human orphan TAS2Rs, we found 9, 17, and 9 ‘new’ agonists for the 3 most broadly tuned human TAS2Rs: TAS2R10, TAS2R14, and TAS2R46, respectively. We also identified additional agonists for TAS2R1 (3), TAS2R4 (2), TAS2R5 (1), TAS2R7 (2), TAS2R8 (1), TAS2R38 (1), TAS2R39 (3), TAS2R40 (2), TAS2R41 (1), TAS2R43 (3), and TAS2R31 (2). Evolutionary relationships of human and mouse bitter taste receptor genes In order to correlate the phylogenetic relationships of mouse and human Tas2rs with their cognate

agonist spectra, we first constructed a phylogenetic tree of all putatively functional mouse and human bitter taste receptors (Figure 7). As pointed out previously (22,23) mouse and human bitter taste receptors do not form separate clusters but rather they intermingle, indicating that ancestral receptor genes existed prior to the divergence of the primate and rodent lineages. Recently, a comprehensive phylogenetic analysis of the bitter taste receptor repertoires in the mammalian superorder Eurarchontoglires, which includes primates and rodents, revealed the existence of 19 one-to-one orthologs among the Tas2r genes of Euarchonta (including primates) and Glires (including rodents) as well as 7 clusters of lineage-specific expansions (56), findings confirming and expanding previous observations (22,23). Due to pseudogenization in either of the grandorders (e.g. TAS2R12, Tas2r208, Tas2r209) putatively functional one-to-one orthologs got lost during evolution leaving 11 of such receptor pairs in the genomes of human and mouse (Figure 7). Focusing on human and mouse bitter taste receptor repertoires, species-specific expansions occurring after the divergence of the Euarchontoglires lineages resulted in the formation of 4 glires- and muroid-specific gene clusters and 1 anthropoidea-specific bitter taste receptor gene cluster (56). The Glires cluster I comprises Tas2r140, Tas2r103, Tas2r129, Tas2r117, Tas2r109, Tas2r123, Tas2r110, Tas2r116, Tas2r113, Tas2r125, and Tas2r115 that together with human TAS2R14 arose from a common ancestor. Muroid cluster I contains Tas2r107, Tas2r106, Tas2r105, Tas2r114, Tas2r104, which together with human TAS2R10 have a common ancestor. Muroid cluster II encompasses Tas2r124, Tas2r102, Tas2r121, and human TAS2R13, which derive from a common ancestor. Moreover, the anthropoid cluster is composed of TAS2R30, TAS2R46, TAS2R45, TAS2R43, TAS2R31, TAS2R19, TAS2R20, TAS2R50, together with mouse Tas2r136 and Tas2r120 (glires cluster II). Role of bitter compounds and their Tas2rs in avoidance behavior In order to examine if the properties of the deorphaned Tas2rs are comparable with or even predict the avoidance behavior of mice, we performed brief-access taste preference tests with a subset of the bitter compounds used in the

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


6

receptor assays. To this end we used C57BL/6 mice, i.e., the same strain from which we cloned the Tas2rs, and substance concentrations equivalent to those used in cell-based assays as well as at least 10-fold lower and higher concentrations. If necessary and within the solubility range, substance concentrations were increased until the animals showed significantly reduced taste preferences relative to water. For the applied compound concentrations, the substance to water lick ratios were monitored (Table 2). The data show that several bitter substances led to receptor activation and reduced substance/water lick ratios with the same potency. Ouabain and D-(-)-salicin decreased lick ratios at 3.0 and 10 mM, respectively, i.e., concentrations that exactly match the threshold concentrations required to stimulate their single cognate receptors, Tas2r144 and Tas2r126 (Table 1 and 2) and consistent with previous observations that mice were indifferent to salicin below 3.0 mM (57). Similar results were obtained for phenanthroline that became aversive at 1.0 mM and activated all of its 5 cognate receptors, Tas2r105, Tas2r121, Tas2r122, Tas2r135, and Tas2r144, with a threshold of 1.0 mM. Moreover, phenyl-β-D-glucopyranoside and chloroquine induced avoidance behavior and stimulated their cognate Tas2rs in a similar concentration range, but both compounds were up to 10-fold less potent in the behavioral assay. Quinine, denatonium benzoate, SOA, thujone, and brucine were avoided at 30-100-fold higher concentrations than those required to activate the cognate receptors in functional expression assays. For acesulfame K and caffeine the difference was several 1000-fold, while PEITC (phenyl ethyl isothiocyanate) did not elicit any avoidance even though its cognate receptor, Tas2r105, became responsive at 0.03 mM. We also investigated avoidance behavior of mice towards 8 substances for which we failed to identify cognate Tas2rs. Of these compounds aloin, amygdalin, atropine, naringin, PTC, and thiamine did elicit aversion at 10-fold higher concentrations than those used in the receptor assays. For berberine and nicotine, the difference was 100-fold. The data suggest that a cognate Tas2r exists for these 8 bitter chemicals but we did not identify them because sufficiently high agonist concentrations could not be employed in the receptor assays.

The data for colchicine differed from the former because it was 100-fold more potent to evoke avoidance than receptor activation. Discussion In our present work we performed a comprehensive analysis of the mouse Tas2r repertoire. In particular, we deorphaned the majority of mouse Tas2rs allowing comparisons of the pharmacological profiles with their well characterized human counterparts as well as with the plethora of behavioral data from previous sensory experiments. Whereas, in general, our results agree well with observations in other species, some findings, such as functional differences among mouse and human bitter taste receptor orthologs, require adjustment of firm believes in light of these data. By in situ hybridization and qRT-PCR experiments we monitored expression of mouse Tas2r genes in taste epithelium and compared their expression levels. Our data indicate that, similar to results obtained previously with human TAS2Rs (58), mouse Tas2rs are indeed all expressed in gustatory tissue confirming a role in bitter taste perception. Moreover, variation in expression levels and numbers agree with the existence of a heterogeneous bitter taste receptor cell population in mouse (9,59). Recently, quantitative expression analyses of rodent Tas2rs have been performed in non-gustatory tissues such as testis and heart (60,61). In mouse testis highest expression was observed for Tas2r113 and Tas2r124, which showed low to moderate expression levels in gustatory tissue (Figure 1A). Moreover, one of the Tas2r genes with lowest expression in lingual papillae, Tas2r114, exhibited robust expression in testis (61). The data suggest that Tas2r gene regulation in taste papillae differs from that in other tissues. Central to this work was the deorphanization and functional characterization of mouse Tas2rs by heterologous expression. We identified agonists for 21 of the 35 putatively functional mouse Tas2rs. The number of identified agonists per receptor revealed that, like human and frog bitter taste receptors (3,17) mouse Tas2rs vary in their tuning breadth. Interestingly, this species displays only a single Tas2r, Tas2r105, that functions as a ‘generalist’ (3), exhibiting an extremely broad agonist profile by recognizing >30% of the bitter

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


7

compound library. Surprisingly, this Tas2r has previously been reported to be highly selective for cycloheximide (7). Of the 24 bitter compounds Chandrashekar and colleagues tested for activation of Tas2r105 (7), we used 19 in the present study and found that in addition to cycloheximide, denatonium, quinine, PROP, and yohimbine also stimulated Tas2r105 transfected cells (Figure 3, 4). This discrepancy appears to be due to differences in experimental methodologies. Heterologous expression analysis of Tas2r105 in HEK293T cells stable expressing Gα15 or Gα16gust44 stimulated with selected agonists, indicated that low efficacy activators of Tas2r105 resulted in lower or even absent responses in Gα15-expressing cells (Figure 5). Therefore, the Gα16gust44 cell system shows higher sensitivity than the Gα15-based assay (7). Mice like humans show similar proportions of moderately tuned Tas2rs responsive to >3% to 10% of the chemicals and of Tas2r ‘specialists’ recognizing less than 3% of the compounds. However, the fact that we discovered activators for only 60% of the mouse Tas2rs, whereas 84% of the human Tas2rs were previously deorphaned with a comparable set of bitter chemicals (3,42), suggests that mice have a higher proportion of specialist receptors relative to humans. For 48 out of 128 compounds we failed to find a sensitive Tas2r and for 13 Tas2rs we were unable to find any bitter agonist. Low receptor expression or a lack of cell surface localization in the heterologous cells as a general cause for the observed failure to identify agonists for these receptors is unlikely because transfection rates, expression levels, and cell membrane localization were not generally correlated between the groups of orphan or deorphaned Tas2rs (Figure 6, Table 3). The inability to deorphan more Tas2rs could be due to non-functional receptor variants generated by single nucleotide polymorphisms in the coding region. Nelson et al. reported that only 2 of 24 Tas2r genes showed no amino acid sequence differences when C57BL/6 and DBA/2J strains were compared (62). These changes in the Tas2r sequences could potentially affect ligand response profiles. Further, a lack of or inefficient G protein coupling might be another confounding feature (41,45,63). The question if animals may recognize rather similar or different arrays of substances eliciting aversive behavior (e.g. bitter taste in human)

cannot be answered conclusively today. Of course, one needs to assume that substances occur in nature that represent relevant toxins for some species and therefore require their recognition by bitter taste receptors, whereas other species may never encounter them and hence, do not rely on receptors detecting these compounds. Answering this question would require the screening of bitter taste receptor repertoires from different species with compound libraries not preselected for their taste in humans. So far, such experiments have not been published and we are aware that by screening mainly substances which taste bitter to humans our compound library has not been unbiased. Indeed, some of the agonists we identified to activate mouse Tas2rs, but failed to activate human TAS2Rs (see below) suggest substantial, but not complete overlap among the bitter taste receptor agonists of both species. Nevertheless, from the bulk of available data, it appears that large overlaps among aversive (bitter) substances exist throughout the animal kingdom. Examples for such overlapping “bitter worlds” are plentiful and extend even to invertebrates possessing phylogenetically unrelated receptors expressed in different (neuronal) cell types. For example the nematode C. elegans shows aversive behavior to the substances quinine, denatonium, and chloroquine (64), substances recognized by human and mouse as well. Such examples of agonist overlaps are even more plentiful for vertebrates ranging from teleostean fish to primates ((17,65-67), this work). Compared to that fewer reports have identified compounds resulting in aversive behavior in other species but fail to activate human bitter taste perception (some compounds presented in this work (see below) and perhaps nicotine for which we have not found a human TAS2R, but identified a chicken Tas2r (17)). Hence, while there is considerable evidence in place for largely overlapping sets of aversive stimuli for many animals ((17,65-67), this work), the assumption of the existence of large groups of species-selective bitter compounds is, even though valid, hypothetical. Nevertheless, we propose that the majority of those Tas2rs that remain orphan represent specialist receptors for compounds that are not contained in our substance library. However, not only the number of agonists differed considerably among the receptors, also the efficacies and potencies of the substances

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


8

interacting with the various mouse receptors deviated. The highest efficacies were observed for cycloheximide (ΔF/F = 1.23 ± 0.20), denatonium saccharide (ΔF/F = 1.06 ± 0.22), and amarogentin (ΔF/F = 0.96 ± 0.24) at Tas2r105, suggesting that this receptor is critical for the recognition of these compounds in vivo when they are present in appropriate concentrations (Figure 3, Supplementary table 3S/A). Other compounds such as the cucurbitacins B, D, E, and I demonstrate more than 10-fold lower efficacy at Tas2r105 (Supplementary table 3S/A). For the recognition of the cucurbitacins in vivo, Tas2r114, may be more relevant because they activate this receptor with substantially higher efficacies. Some substances, such as diphenidol and phenanthroline, activated their cognate receptors with similar efficacies. Thus, their overall bitterness is less likely to be dependent on a single Tas2r. The potencies of bitter compounds also deviated largely across compounds for the same as well as different Tas2rs (Table 1, Supplementary table 3S/B). The highest potency with a threshold concentration of 10 nM and an EC50-concentration of 0.3 ± 0.2 µM was observed for cycloheximide at Tas2r105 (Figure 3), confirming the dominant role of this receptor for the exquisite cycloheximide sensitivity of mice (7). Other agonists activated the receptor with at least ~10-fold lower potencies, together spanning a concentration range of about 4 orders of magnitude. Most substances showed very low potencies in the millimolar concentration range for their cognate Tas2rs. One of these substances is PROP, which activated 6 receptors at thresholds of only 0.3 to 1.0 mM (Table 1). Thereby, Tas2r138 did not respond to PROP whereas the orthologous human receptor, TAS2R38, is exquisitely sensitive to PROP showing an EC50-concentration of 2.1 µM (33). The β-D-glucopyranosides arbutin, helicin, phenyl-β-D-glucopyranoside, and D-(-)-salicin all activate the receptor Tas2r126 with the highest observed threshold concentrations between 10 and 30 mM. However, in contrast to human TAS2R16 (34), the mouse Tas2r126 recognition pattern is not limited to β-D-glucopyranosides. Hence, mice appear to lack Tas2rs detecting common structural configurations such as human TAS2R16 and TAS2R38. In some cases compounds activating multiple Tas2rs displayed similar potencies. For example,

the 7 receptors Tas2r105, Tas2r108, Tas2r115, Tas2r126, Tas2r137, Tas2r140, and Tas2r144 are activated by quinine at concentrations between 3.0 and 10 µM. However, for other compounds the concentrations required to activate different Tas2rs are staggered. A good example for this is the artificial sweetener saccharin which activates Tas2r135, Tas2r105, Tas2r109, and Tas2r144 with threshold concentrations of 0.1, 1.0, 3.0, and 10 mM, respectively. Hence, it is conceivable to assume that increasing concentrations of saccharin in vivo results in a graded bitter response involving 1 to 4 Tas2rs. In total, like human TAS2Rs (3), mouse Tas2rs display threshold concentrations for bitter chemicals spanning 6 orders of magnitude. The results of the present study allow a systematic comparison of the agonist spectra of mouse and human bitter taste receptors. To provide an even broader basis for such comparisons we subjected human TAS2Rs to a screening with numerous substances not tested before. These new agonist data did not change the classification of human TAS2Rs in broadly, moderately, or narrowly tuned receptors (3). Our data further revealed that mice and humans detect a similar set of bitter compounds. Out of the 128 substances used to challenge both, the mouse and human Tas2rs, 80 (63%) activated mouse Tas2rs and 98 (77%) human TAS2Rs of which 72 substances (56%) stimulated Tas2rs in both species. We identified 8 compounds that were selective for mouse Tas2rs (Table 1, shown in bold), whereas 26 substances specifically stimulated human TAS2Rs ((3) and supplementary table 3S C). Twenty two (17%) test substances did neither activate mouse nor human Tas2rs probably because higher concentrations would be required to evoke Tas2r responses. The ability of individual bitter compounds to activate multiple bitter taste receptors also varied between mouse and humans. Whereas quinine, activated similar numbers of mouse (7 receptors) and human TAS2Rs (9 receptors), diphenidol stimulated more than twice as many Tas2rs in humans (15 receptors) than in mice (6 receptors) (3). Vice versa, other substances such as PROP, with one main receptor in human, the TAS2R38, acts more broadly on mouse Tas2rs being an agonist for 6 receptors. Thus, the response patterns of mouse and human bitter taste receptors are heterogeneous.

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


9

An important question is if one-to-one orthologous Tas2rs represent functional orthologs, e.g. show identical or at least similar agonist profiles? Whereas 4 one-to-one orthologous pairs cannot be compared because either the human, mouse, or both Tas2rs remained orphan, for 7 orthologous pairs this comparison was possible (Figure 8A). Of these receptor pairs, Tas2r108 and its human ortholog TAS2R4 exhibit the highest degree of overlap in their set of agonists. Of the 18 bitter compounds for this pair, we observed that a third activated both receptors. The substances capable of activating both receptors do not show apparent structural similarities. Only one out of 12 bitter substances is commonly recognized by Tas2r138 and TAS2R38. It is remarkable that the two prototypical agonists for the human TAS2R38, PROP and PTC (phenylthiocarbamide), are not activators of the orthologous mouse receptor, Tas2r138, which has been frequently, but erroneously, assumed in the past (25-28). Whereas in human the sensitivities for PROP and PTC are highly correlated to their activation of TAS2R38 (33,38), they are not in mice, suggesting that different mouse Tas2rs are underlying responsiveness to these substances (68). In fact, a polygenic control of PROP sensitivity was suggested (69), which is in good agreement with our observation that PROP activated 6 Tas2rs (Table 1). Other pairs of orthologs share few or even not a single agonist (Figure 8A) demonstrating that, in general, orthologous Tas2rs have largely distinct agonist profiles. The little overlap is probably at chance level and is not unexpected given the broad tuning of Tas2rs. In fact, statistical analyses (fourfold X2-test) confirmed that the number of common agonists for all but one pair (TAS2R4/Tas2r108) of the one-to-one orthologs did not exceed chance levels. While we cannot exclude that additional common bitter agonists for these one-to-one orthologous receptor pairs exist in nature, it seems that also these receptors contribute to species-specific bitter substance recognition. A good example for this is the human TAS2R38 (70,71). Comparing data from structure-function analyses, amino acid sequence homologies, and pharmacological properties of TAS2R38 orthologues, suggests that the receptor was modified differently during the evolution of Euarchontoglires, a clade including primates and

rodents (cf. (56)). While primate TAS2R38 acquired sensitive PTC responsiveness as well as activation by PROP, this was not the case for the rodent ortholog Tas2r138 (Figure 9, upper panels). A comparison of functionally critical residues in selected TAS2R38 orthologs of the Euarchontoglires clade revealed that all invariantly exhibit amino acid residues characteristic for the human taster variant, TAS2R38-PAV, or a variation thereof (TAS2R38-PAI), which has been experimentally validated for full PTC/PROP-responsiveness (33) (Figure 9, lower panel). Previous in vitro mutagenesis experiments combined with functional heterologous expression assays have revealed several functionally important residues in the binding pocket of human TAS2R38 contributing to PROP and PTC activation (70,71). Among these residues were tryptophane 201 (5.46), serine 260 (6.52), and phenylalanine 264 (6.56). With one exception, the mutation of serine 260 to alanine which resulted in unimpaired responsiveness to PROP, but not PTC, all modifications resulted in severely reduced activation of the mutated receptors (71). In particular, exchanging tryptophane in position 201 for leucine or phenylalanine caused severely reduced PTC responsiveness and, practically, a loss of activation by PROP. Intriguingly, Trp5.46 is only found in the haplorrhine primate clade including human and chimpanzee, which exhibit exquisitely PTC- as well as PROP-sensitive TAS2R38 receptors (33,66). Hence, it seems that PTC/PROP-sensitive TAS2R38 evolved within the Primate order in the haplorrhine branch. Moreover, also the residue at position 6.52 showed a strict separation among the compared clades. In this case, all but the Catarrhini, which carry a serine residue at this position, have a phenylalanine (or valine in case of Jaculus jaculus belonging to Jerboa) at this position. Strikingly, this position as well affected PTC and PROP recognition in vitro with serine being the preferential residue for the activation by both substances (71). Finally, position 6.56 differs among Catarrhini, which exhibit a phenylalanine residue, and the other species with different, dominantly hydrophobic residues at this position. As experimental evidence suggests the requirement of phenylalanine at this position for full PTC/PROP responsiveness and rodent receptors differ in all 3 mentioned positions from

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


10

the human and chimpanzee counterparts, we conclude that this is the underlying reason for the fact that the mouse receptor and perhaps all rodent receptors are not capable of interacting with PTC or PROP. This indicates that functional divergence has occurred before separation of rodent and primate lineages at the beginning of the earlier Cretaceous period. Nevertheless, since the cat Tas2r38 ortholog shows insensitive PTC responsiveness and no PROP responses (72,73) we assume the existence of a common ancestral TAS2R38 ortholog permissive for PTC/PROP-responses. Of course, the existence of further critical positions for PTC/PROP-responses cannot be ruled out and may contribute significantly. This example indicates that pharmacological diversification occurs also among the group of one-to-one orthologous receptors. Therefore, the hypothesis that orthologous receptors may recognize bitter compounds important for both species (23) seems, at least for most cases, not to be true. The persistence of these genes intact in the genomes of mice and humans and not pseudogenized could indicate that common, yet still unknown, bitter substances which pose or have posed a severe threat to the survival of both organisms throughout evolution could exist for these one-to-one orthologs. Alternatively, the corresponding receptors may fulfill another and dominant function beyond bitter taste perception by recognizing endogenous and well conserved agonists. As the number of reports on extragustatory expression of bitter taste receptors is ever increasing (24,44), this hypothesis does not seem to be too farfetched. Lastly, the structure of these receptors may have allowed the rapid evolution of binding sites tailored to recognize compounds for the specific needs of mice or humans. In that case, the unique ability of bitter taste receptors to dynamically adapt their functions to the nutritional requirements of organisms might be more important than the fixation of pharmacological properties. In contrast to the one-to-one orthologous receptor pairs, it is assumed that lineage-specific expansions possibly generated Tas2rs critical for the recognition of bitter substances that are encountered only in the concerned species (23). If this were true, then the cluster of amplified Tas2rs in one species should recognize more compounds

than the related single Tas2r in the other species, some of which should be species-specific. Glires cluster I consists of 11 mouse Tas2rs and human TAS2R14, which is the most broadly tuned TAS2R in humans. In the course of our experiments, we found agonists for 8 of the 11 mouse Tas2rs, while 3 remained orphan. Of the 64 agonists activating the Tas2rs of Glires cluster I the majority, namely 38, were specific for human TAS2R14, 11 activated the human TAS2R14 and several mouse Tas2rs, whereas only 15 substances were specific for the mouse Tas2rs of this group. For muroid cluster I, which includes one broadly tuned human receptor together with 5 mouse Tas2rs, of which we deorphaned 2 receptors, as well as muroid cluster II with 1 human TAS2R and 3 mouse Tas2rs we found similar results (Figure 8B). In contrast to clusters showing an expansion of mouse Tas2rs, in Glires cluster II/anthropoid cluster, the mouse paralogs Tas2r136 and Tas2r120 go together with 8 human TAS2Rs. Of these receptors, mouse Tas2r136 as well as human TAS2R19 and TAS2R45 are orphan receptors and cannot be compared. Only a single compound out of the 54 activators for this cluster was specific for a murine Tas2r and 2 substances activated Tas2rs in both species (Figure 8B). Remarkably, the remaining 51 chemicals were selective for the human TAS2Rs of this cluster. Thus, the hypothesis that the lineage-specific expansions generated Tas2rs for species-specific bitter chemicals is not generally supported by our data. For example, only 5 out of 26 human-specific compounds are recognized by members of anthropoid cluster, whereas most of the human-specific compounds are recognized by the 3 most broadly tuned receptors: TAS2R10, TAS2R14, and TAS2R46. However, the bitter taste receptor gene expansions contribute to a broadening of the overall agonist profiles which may be particularly important for the more narrowly tuned mouse receptors and hence, may account for the fact that more frequent gene expansions occurred in mice. In fact, a closer look on the amino acid sequence of receptors of murine cluster I show a general tendency of higher amino acid sequence homologies in intracellularly oriented transmembrane domain (TM) parts and intracellular loops (ICL) compared to extracellular TM regions and extracellular loops (ECL), which has been recognized before (10). A detailed

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


11

comparative analysis of receptor positions constituting the binding pockets of murine cluster I receptors suggest that diversification of agonist spectra has occurred (Figure 10). All receptors of murine cluster 1 containing human TAS2R10, which has been subjected to detailed structure-function analyses (30), exhibit a different combination of amino acid residues at positions showing pronounced agonist selectivity and hence, their corresponding putative agonist spectrum. The concentration-response relationships for selected groups of human and mouse Tas2rs indicate that the activation properties of sequence related human and mouse Tas2rs differ more substantially than is evident from sole comparisons of their agonist profiles (Figure 11). Sequence orthologs such as TAS2R38/Tas2r138 or TAS2R1/Tas2r119 can or cannot recognize the same compounds (Figure 11A, B). However, even if they do, potencies and efficacies differ substantially (Figure 11B). The same pronounced differences are also seen in the case of members of muroid cluster I (Figure 11C, D) or of representatives of Glires cluster II/anthropoid cluster (Figure 11E, F). Another hypothesis about the development of species-specific Tas2r gene clusters concerns tuning breadth rather than individual agonist spectra (3). Because both of the single human TAS2Rs corresponding to mouse Tas2r gene clusters (Glires cluster I and muroid cluster I) are extraordinarily broadly tuned receptors, one could assume that the gene expansion in the rodent lineage resulted in the development of multiple specialized receptors arising from a broadly tuned ancestral receptor or that the broad tuning of the ancestral receptor was maintained by the derived Tas2rs to cover an even larger chemical space. Our results strongly suggest that multiple broadly tuned receptors were not generated, but rather a specification of several receptors occurred (Table 1). Our analyses of bitter compounds and the corresponding mouse Tas2r for avoidance behavior revealed that in some but not in all cases the sensitivity of Tas2r responses measured in vitro matched the concentration range of the substance in vitro. Whether these differences could be due to perireceptor events (74) not mimicked in our in vitro assays remains to be determined. The data suggest that receptor

threshold values can in some cases predict bitterness avoidance of mice. However, it appears that for other bitter chemicals other possible factors such as the interaction with the oral mucosa or saliva may reduce their potency of inducing aversion. However, in view of different G protein coupling of Tas2rs in vitro and in vivo (41,45,63,75,76), the data of the behavioral experiments agree reasonably well with the data from the receptor assays on a qualitative level and in some cases also on a quantitative level. In case of the substance colchicine, which showed a 100-fold higher potency when eliciting avoidance behavior in vivo compared to the receptor assays, other explanations need to be taken into account. Either the ‘best’ receptor has not been discovered or alternative recognition mechanisms exist that do not rely on Tas2rs. However, the ability of colchicine to activate three human, one chicken, one turkey, and one frog receptor (17) suggests that Tas2r-dependent detection mechanisms likely exist for this compound. Molecular genetics can shed light on the importance of specific Tas2rs for taste-relevant behavior. The importance of Tas2r105 for cycloheximide recognition is illuminated by strain-specific differences leading to the identification of a chromosomal locus mediating cycloheximide sensitivity (51) and in Tas2r105 knockout mice, which demonstrated loss of nerve responses and behavioral aversion to this translational inhibitor (57). However, avoidance of denatonium, PROP, and quinine was not altered in these mice. Since all three substances activate four to six other Tas2rs, it is conceivable that they suffice to evoke avoidance of those compounds. Strain-specific recognition extends to other bitter compounds, leading to the identification of chromosomal regions critical for the detection of quinine (Qui; (49,68,69,77) or Soa (48,78,79), yet the cognate Tas2r genes remained unknown. The close genetic linkage of SOA and strychnine sensitivity (80) agrees well with our finding that only a single receptor, Tas2r117, responds to both compounds. Intriguingly, the Tas2r117 sequence is fully intact in C57BL/6 mice used for our analyses, but contains missense mutations and small deletions in the DBA/2J strain (62), likely explaining the inability of DBA/2J mice to taste SOA, strychnine, and brucine (47,80,81), which are all activators of Tas2r117.

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


12

Recently, it was reported that solitary chemosensory cells (SCC) in the anterior nasal epithelium express Tas2rs and their signaling molecules (82) respond to bacterial quorum-sensing AHLs (82,83). Our experiments revealed that mouse Tas2r105, as well as human TAS2R1, TAS2R10, and TAS2R14, were sensitive to various AHLs. We failed, however, to monitor AHL responses for TAS2R38 (55), which may be explained by the use of different experimental methodologies. The ability to detect quorum sensing molecules contributes to environmental adaptions and influences the behavior of eukaryotic organisms (84,85). Humans homozygous for the non-taster allele of TAS2R38 are reported to be more susceptible to upper respiratory tract infections by Gram-negative bacteria than individuals carrying the taster-variant of this receptor (55). It would be interesting to see if this also applies to those strains of mice harboring the less sensitive variant of Tas2r105 (7). In light of findings showing that Tas2rs are present in organs that are not or only partially accessible to xenobiotics such as brain (86-89), testes (61,90,91), thyroid (92), and urethra (93), we also examined if hormones could function as Tas2r activators. We found that progesterone stimulated Tas2r114 and Tas2r110. This steroid hormone is expressed in ovaries (corpus luteum), the adrenal glands, and testicular Leydig-cells. It also has major effects on human sperm motility (94). While Tas2r114 is expressed in gustatory tissue at rather low levels, its RNA is abundantly present in testis (61). Mouse spermatids and spermatozoa respond to bitter compounds with calcium signaling in a α-gustducin dependent manner (61). Genetic ablation of bitter receptor cells in mice caused massive spermatid loss (90). Further studies are needed to elucidate the role of this and other Tas2rs in testicular function. Taken together the work presented here sheds light on the evolutionary dynamics that acted on the bitter receptor repertoires of vertebrates, which resulted in the development of highly versatile GPCRs capable to adapt to various lifestyles and habitats. Experimental procedures Mice

Animal care and procedures were performed following national and institutional guidelines, approved by the local animal experimentation committee (Ministry of Environment, Health and Consumer Protection Brandenburg, Germany; V3-2347-01-2013; 23-2347-A-1-1-2010). Mice were housed in polycarbonate cages and kept under 12h/12h light/dark cycle unless otherwise noted with water and food ad libitum. Brief-access tests Behavioral studies with C57BL/6 mice (n = 8; male, 8- to 9-week-old) obtained from Janvier (Le Genest St. Isle, France) were carried out using a Davis rig MS-160 (DiLog Instruments, Tallahassee, USA). Prior to testing, animals were housed individually with food available ad libitum. Mice were water-deprived for 18 h for the first three days of adaptation, followed by restriction periods of 22.5 h in preparation for taste solution presentation. Each stimulus and concentration was presented in three independent test sessions in 5 s-trials. Inter-trial intervals were 7.5 s during a 20 min session as reported before (68,95-98). For detailed information about substances see Table 2 and Supplementary table 2S. Statistical significance was determined by analysis of mixed models and post-hoc-analysis using Bonferroni-test (SPSS 16.0, IBM, New York, USA). Tissue preparation Lingual epithelium was isolated from mice that were anesthetized using isoflurane (Baxter, Vienna, Austria) and sacrificed by cervical dislocation. Epithelium was removed as published earlier (91). The peeled tissue was fixed in a Sylgard dish and epithelium containing taste papillae was dissected from surrounding tissue. Papillae-enriched epithelium was frozen at -80°C and finally used for RNA extraction (see quantitative RT-PCR). To prepare fixed tissue, C57BL/6 mice were anesthetized with 150 mg/kg body weight Narcoren (Merial, Hallbergmoos, Germany) and transcardially perfused with phosphate buffered saline (PBS) followed by ice-cold 4% paraformaldehyde (PFA). Tongues were removed, postfixed for 2 h in 4% PFA and soaked in 30% sucrose overnight at 4°C. Tissues were sectioned

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


13

on cryostate (10 µM) and thaw-mounted onto Superfrost Plus slides (Menzel, Roth, Germany). Quantitative RT-PCR Epithelial preparations enriched in vallate and foliate papillae were subjected to RNA extraction using TRIzol reagent (Invitrogen, Karlsruhe, Germany). Following DNAse I (Invitrogen) digestion to remove potentially contaminating genomic DNA, cDNA was synthesized using SuperScript III (Invitrogen) according to the manufacturer’s instructions. Gene specific primers and TaqMan probes (Supplementary table 1S) were used to amplify Tas2r and α-gustducin RNA. The mRNA for β-actin served as internal control. Quantitative analysis was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems, Darmstadt, Germany). For PCR reactions cDNA corresponding to 6.25 ng total RNA was mixed with 0.5 µM probe, 1.25 µM of each primer, and 1x TaqMan ® Gene Expression Master Mix (Applied Biosystems). The following cycles were run: 50°C for 2 min, 95°C for 10 min; followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Each cDNA sample was run in triplicates. For negative controls corresponding samples without prior reverse transcriptase reaction and water controls were used. Threshold cycle (CT) values were acquired with 7500 Software v2.0.1 (Applied Biosystems). Mean values of CT triplets were calculated (single values differing ≥1 CT were excluded), and ΔCT values were determined using mean CT of β-actin as reference (ΔCT = CT target – CT reference). Finally, 2-ΔC

T values were calculated based on established methods (99). In situ hybridization Coding sequences of Tas2rs were subcloned into vectors with N-terminal rat somatostatin receptor subtype 3 (sst3) tag and a C-terminally located herpes simplex virus glycoprotein D epitope (HSV; see calcium imaging analyses for more details). Using primers specific for the coding sequences of sst3 and HSV tags cDNAs containing T3- and T7-RNA-polymerase promoter sequences were amplified and subsequently used as template for in vitro transcription reactions as described before (58). All probes were hybridized to template plasmid DNA spotted onto nitrocellulose membranes to adjust sense and

antisense probes for similar detection efficiencies. In situ hybridization was done as described previously (91) with 10 µm tissue sections of vallate papillae from C57BL/6 mice. Taste compounds All analyzed compounds have previously been described in the literature to taste bitter (100-105) or represent small molecules responsible for cell-to-cell communications that have been conjectured to activate bitter taste receptors outside the oral cavity (55,106). Bitter chemicals were mainly purchased from Sigma-Aldrich (Taufkirchen, Germany), Fluka (Oberhaching, Germany), ABCR (Karlsruhe, Germany), ICN Biochemicals (Aurora, USA), BioChemica (Darmstadt, Germany), ChromaDex (LGC Standard, Wesel, Germany), CPS Chemie+Service GmbH (Aachen, Germany), Cfm Oskar Tropitzsch (Marktredwitz, Germany), Carl Roth (Karlsruhe, Germany), and HWI Analytik (Rülzheim, Germany). Homoserine lactones were acquired from Darren Furniss (University of Nottingham, United Kingdom). Non-commercial bitter terpenoids were obtained by isolation from their plant source (107-112). All other substances were available from previous studies (32,37). For detailed information about manufacturers and applied concentrations see Supplementary table 2S. Test compounds for calcium imaging experiments were dissolved directly in C1 buffer (130 mM NaCl, 5 mM KCl, 10 mM Hepes, 2 mM CaCl2, 10 mM glucose, pH 7.4) or dimethyl sulfoxide (DMSO) solution. Dimethyl sulfoxide stock solutions were further diluted in C1 buffer, not exceeding a final DMSO concentration of 1%. For behavioral studies, taste solutions were prepared in distilled water freshly every day and presented at room temperature. Substances were identical to the ones used in functional expression analyses with exception of quinine, which was applied as quinine hydrochloride for behavioral studies (Table 2). Calcium imaging analysis Coding sequences of mouse bitter taste receptors (excluding stop codon; for accession number see Supplementary table 1S) were amplified from genomic DNA of C57BL/6 animals and subcloned into expression plasmids based on pcDNA5/FRT (Invitrogen; Tas2r105, Tas2r114, Tas2r119,

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


14

Tas2r138, Tas2r143, and Tas2r144) or pEAK10 (Edge BioSystems, Gaithersburg, USA; all other receptors; (113)). Receptor sequences correspond to published data (NCBI; 2013; for accession number see Supplementary table 1S) with one exception: Sequence of Tas2r105 is identical to that contained in GenBank except a synonymous CCA to CCG transition at position 219. The Tas2r coding sequences in the expression plasmids are flanked at their amino termini by the first 45 amino acids of the rat somatostatin receptor subtype 3 (114) to improve plasma membrane targeting and by the HSV epitope at their carboxyl termini the for immunohistochemical detection (34). Despite substantial cloning effort, generation of Tas2r116 was not possible. Functional expression analysis was performed as described earlier for human, frog, and chicken bitter taste receptors (e. g. (3,17,29,32,33,115)). Briefly, receptor cDNA constructs were transiently transfected in human embryonic kidney (HEK)-293T cells stably expressing the chimeric G protein Gα16gust44 using Lipofectamine 2000 (Invitrogen) (33,115,116). Twenty-three to 26 hours later cells were loaded with Fluo-4 AM (Molecular Probes, Karlsruhe, Germany) in the presence of probenecid (2.5 mM; Sigma-Aldrich) in serum-free medium. Next, loading solution was removed and cells were incubated in C1 solution until the beginning of the experiment. For functional expression analyses taste solutions were automatically applied to the cells. To avoid false positive signals, substances were screened at concentrations not eliciting artificial signals in empty vector (mock)-transfected cells (supplementary table 2S), which were used as negative controls. To demonstrate cell vitality a second application with a final concentration of 100 nM somatostatin-14 (Bachem, Weil am Rhein, Germany) was included to activate endogenous somatostatin receptors. Data were collected from at least two independent experiments carried out in triplicates. Calcium responses were base line-corrected and expressed as ΔF/F by subtracting fluorescence changes of mock-transfected cells from those of receptor-transfected cells. For dose-response curves signals, two to four experiments were averaged and the fluorescence changes of corresponding mock-transfected cells were subtracted. Signals were normalized to background fluorescence. For the calculation of

EC50-values plots of amplitude versus log concentrations were prepared in Sigma Plot 11.0 (SPSS Inc., IBM) by non-linear regression of the plots using the function f (x) = (a-d)/1+[(x/EC50)

nH]+d; with a = max, d = min, x = agonist concentration and nH = Hill coefficient. The lowest concentration generating a calcium signal was specified as threshold concentration. Immunocytochemisty To determine the cellular expression levels of mouse Tas2rs and their cell surface localization selected receptor coding sequences (excluding the stop codon) were cloned in pcDNA5/FRT expression vector, resulting in Tas2r sequences flanked by the first 60 amino acids of rhodopsin (Rho-tag) (117), which can be detected with suitable antisera in vivo (118), and HSV-tag epitope at the amino and carboxyl termini, respectively. HEK293T-Gα16gust44 cells were grown and transfected with Rho-tagged receptor plasmids on poly-D-lysine-coated glass coverslips as published before (e.g. (1,30,31)). To visualize the cells, their surfaces were stained with 5 µg/ml biotin-conjugated concanavalin A (Sigma-Aldrich) and streptavidin-conjugated Alexa Fluor 633 (1:1,000; Molecular Probes). The Rho-tagged receptors were visualized by staining with an anti-Rho primary antibody (1:500; Abcam, Rho 4D2, Cambridge, UK) and an Alexa Fluor 488-labeled goat anti-mouse secondary antibody (1:2,000; Molecular Probes). Rho-antibody was either applied before or after fixation to the cells in 3% normal horse serum in 1x PBS and left for 1 h on ice (if added prior to fixation) or at room temperature (if added after fixation), respectively. Fixation was done by acetone/methanol (1:1, v/v) treatment for 2 min and subsequent washing with 1x PBS. Receptor expression was recorded using the confocal laser-scanning microscope Leica TCS-SP8 (Leica, Wetzlar, Germany). Phylogenetic tree Coding nucleotide sequences of mouse Tas2r genes were obtained from the genomic scaffolds NC_000068.7, NC_000072.6, and NC_000081.6 of the mouse genome assembly GRCm38.p2 released by the Genome Reference Consortium (119). Coding nucleotide sequences of human TAS2R genes were obtained from the genomic scaffolds 1103279188109, 1103279188381,

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


15

1103279188228, and 1103279188408 of the whole genome assembly released by the Venter Institute as Human Reference genome (120). A global multiple sequence alignment was performed for amino acid sequences corresponding to mouse and human bitter taste receptor genes using a modified version of the Feng-Doolittle (121) progressive alignment

algorithm (Align X, Vector NTI; Life Technologies, Carlsbad, CA, USA). A neighbor-joining tree (Clustal X; (122)) was constructed after manual adjustment whilst considering previous in silico and in vitro experiments (29,30). Bootstrap values computed using 10,000 iterations were subsequently inferred.

Acknowledgements The authors thank Elke Chudoba, Josefine Würfel, Florian Padberg, Lisa Oldorff, Julia Freydank, Alexandra Semmler, and Eva-Katharina Hage (Nuthetal) for expert technical assistance. Conflict of interest Jay P. Slack is employee of Givaudan Flavors Corp. The other authors have nothing to disclose. Author contributions KL designed research, constructed vectors for functional expression, performed calcium imaging analysis, immunocytochemistry, and brief-access tests, and prepared the manuscript. SH carried out qRT-PCR, prepared in situ hybridization probes and performed corresponding experiments. NR conducted phylogenetic tree and statistical analysis. JPS contributed to the design of the study. FP isolated and/or repurified several sequiterpene lactones. MB and MW designed research and prepared the manuscript. All authors read and approved of the final version of the manuscript. References 1. Behrens, M., Brockhoff, A., Kuhn, C., Bufe, B., Winnig, M., and Meyerhof, W. (2004) The

human taste receptor hTAS2R14 responds to a variety of different bitter compounds. Biochem Biophys Res Commun 319, 479-485

2. Roland, W. S., Vincken, J. P., Gouka, R. J., van Buren, L., Gruppen, H., and Smit, G. (2011) Soy isoflavones and other isoflavonoids activate the human bitter taste receptors hTAS2R14 and hTAS2R39. J Agric Food Chem 59, 11764-11771

3. Meyerhof, W., Batram, C., Kuhn, C., Brockhoff, A., Chudoba, E., Bufe, B., Appendino, G., and Behrens, M. (2010) The molecular receptive ranges of human TAS2R bitter taste receptors. Chem Senses 35, 157-170

4. Soares, S., Kohl, S., Thalmann, S., Mateus, N., Meyerhof, W., and De Freitas, V. (2013) Different phenolic compounds activate distinct human bitter taste receptors. J Agric Food Chem 61, 1525-1533

5. Behrens, M., and Meyerhof, W. (2013) Bitter taste receptor research comes of age: from characterization to modulation of TAS2Rs. Semin Cell Dev Biol 24, 215-221

6. Adler, E., Hoon, M. A., Mueller, K. L., Chandrashekar, J., Ryba, N. J., and Zuker, C. S. (2000) A novel family of mammalian taste receptors. Cell 100, 693-702

7. Chandrashekar, J., Mueller, K. L., Hoon, M. A., Adler, E., Feng, L., Guo, W., Zuker, C. S., and Ryba, N. J. (2000) T2Rs function as bitter taste receptors. Cell 100, 703-711

8. Fredriksson, R., Lagerstrom, M. C., Lundin, L. G., and Schioth, H. B. (2003) The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol Pharmacol 63, 1256-1272

9. Matsunami, H., Montmayeur, J. P., and Buck, L. B. (2000) A family of candidate taste receptors in human and mouse. Nature 404, 601-604

10. Meyerhof, W. (2005) Elucidation of mammalian bitter taste. Rev Physiol Biochem Pharmacol 154, 37-72

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


16

11. Grus, W. E., and Zhang, J. (2009) Origin of the genetic components of the vomeronasal system in the common ancestor of all extant vertebrates. Mol Biol Evol 26, 407-419

12. Dong, D., Jones, G., and Zhang, S. (2009) Dynamic evolution of bitter taste receptor genes in vertebrates. BMC Evol Biol 9, 12

13. Jiang, P., Josue, J., Li, X., Glaser, D., Li, W., Brand, J. G., Margolskee, R. F., Reed, D. R., and Beauchamp, G. K. (2012) Major taste loss in carnivorous mammals. Proc Natl Acad Sci U S A 109, 4956-4961

14. Zhao, H., Li, J., and Zhang, J. (2015) Molecular evidence for the loss of three basic tastes in penguins. Curr Biol 25, R141-142

15. Zhu, K., Zhou, X., Xu, S., Sun, D., Ren, W., Zhou, K., and Yang, G. (2014) The loss of taste genes in cetaceans. BMC Evol Biol 14, 218

16. Feng, P., Zheng, J., Rossiter, S. J., Wang, D., and Zhao, H. (2014) Massive losses of taste receptor genes in toothed and baleen whales. Genome Biol Evol 6, 1254-1265

17. Behrens, M., Korsching, S. I., and Meyerhof, W. (2014) Tuning properties of avian and frog bitter taste receptors dynamically fit gene repertoire sizes. Mol Biol Evol 31, 3216-3227

18. Li, D., and Zhang, J. (2014) Diet shapes the evolution of the vertebrate bitter taste receptor gene repertoire. Mol Biol Evol 31, 303-309

19. Shi, P., and Zhang, J. (2006) Contrasting modes of evolution between vertebrate sweet/umami receptor genes and bitter receptor genes. Mol Biol Evol 23, 292-300

20. Syed, A. S., and Korsching, S. I. (2014) Positive Darwinian selection in the singularly large taste receptor gene family of an 'ancient' fish, Latimeria chalumnae. BMC Genomics 15, 650

21. Go, Y., Satta, Y., Takenaka, O., and Takahata, N. (2005) Lineage-specific loss of function of bitter taste receptor genes in humans and nonhuman primates. Genetics 170, 313-326

22. Conte, C., Ebeling, M., Marcuz, A., Nef, P., and Andres-Barquin, P. J. (2003) Evolutionary relationships of the Tas2r receptor gene families in mouse and human. Physiol Genomics 14, 73-82

23. Shi, P., Zhang, J., Yang, H., and Zhang, Y. P. (2003) Adaptive diversification of bitter taste receptor genes in Mammalian evolution. Mol Biol Evol 20, 805-814

24. Behrens, M., and Meyerhof, W. (2011) Gustatory and extragustatory functions of mammalian taste receptors. Physiol Behav 105, 4-13

25. Chen, M. C., Wu, S. V., Reeve, J. R., Jr., and Rozengurt, E. (2006) Bitter stimuli induce Ca2+ signaling and CCK release in enteroendocrine STC-1 cells: role of L-type voltage-sensitive Ca2+ channels. Am J Physiol Cell Physiol 291, C726-739

26. Janssen, S., Laermans, J., Verhulst, P. J., Thijs, T., Tack, J., and Depoortere, I. (2011) Bitter taste receptors and alpha-gustducin regulate the secretion of ghrelin with functional effects on food intake and gastric emptying. Proc Natl Acad Sci U S A 108, 2094-2099

27. Jeon, T. I., Zhu, B., Larson, J. L., and Osborne, T. F. (2008) SREBP-2 regulates gut peptide secretion through intestinal bitter taste receptor signaling in mice. J Clin Invest 118, 3693-3700

28. Wu, S. V., Chen, M. C., and Rozengurt, E. (2005) Genomic organization, expression, and function of bitter taste receptors (T2R) in mouse and rat. Physiol Genomics 22, 139-149

29. Brockhoff, A., Behrens, M., Niv, M. Y., and Meyerhof, W. (2010) Structural requirements of bitter taste receptor activation. Proc Natl Acad Sci U S A 107, 11110-11115

30. Born, S., Levit, A., Niv, M. Y., Meyerhof, W., and Behrens, M. (2013) The human bitter taste receptor TAS2R10 is tailored to accommodate numerous diverse ligands. J Neurosci 33, 201-213

31. Behrens, M., Brockhoff, A., Batram, C., Kuhn, C., Appendino, G., and Meyerhof, W. (2009) The human bitter taste receptor hTAS2R50 is activated by the two natural bitter terpenoids andrographolide and amarogentin. J Agric Food Chem 57, 9860-9866

32. Brockhoff, A., Behrens, M., Massarotti, A., Appendino, G., and Meyerhof, W. (2007) Broad tuning of the human bitter taste receptor hTAS2R46 to various sesquiterpene lactones, clerodane and labdane diterpenoids, strychnine, and denatonium. J Agric Food Chem 55, 6236-6243

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


17

33. Bufe, B., Breslin, P. A., Kuhn, C., Reed, D. R., Tharp, C. D., Slack, J. P., Kim, U. K., Drayna, D., and Meyerhof, W. (2005) The molecular basis of individual differences in phenylthiocarbamide and propylthiouracil bitterness perception. Curr Biol 15, 322-327

34. Bufe, B., Hofmann, T., Krautwurst, D., Raguse, J. D., and Meyerhof, W. (2002) The human TAS2R16 receptor mediates bitter taste in response to beta-glucopyranosides. Nat Genet 32, 397-401

35. Kuhn, C., Bufe, B., Winnig, M., Hofmann, T., Frank, O., Behrens, M., Lewtschenko, T., Slack, J. P., Ward, C. D., and Meyerhof, W. (2004) Bitter taste receptors for saccharin and acesulfame K. J Neurosci 24, 10260-10265

36. Dotson, C. D., Zhang, L., Xu, H., Shin, Y. K., Vigues, S., Ott, S. H., Elson, A. E., Choi, H. J., Shaw, H., Egan, J. M., Mitchell, B. D., Li, X., Steinle, N. I., and Munger, S. D. (2008) Bitter taste receptors influence glucose homeostasis. PLoS One 3, e3974

37. Intelmann, D., Batram, C., Kuhn, C., Haseleu, G., Meyerhof, W., and Hofmann, T. (2009) Three TAS2R Bitter Taste Receptors Mediate the Psychophysical Responses to Bitter Compounds of Hops (Humulus lupulus L.) and Beer. Chemosensory Perception 2, 118-132

38. Kim, U. K., Jorgenson, E., Coon, H., Leppert, M., Risch, N., and Drayna, D. (2003) Positional cloning of the human quantitative trait locus underlying taste sensitivity to phenylthiocarbamide. Science 299, 1221-1225

39. Maehashi, K., Matano, M., Wang, H., Vo, L. A., Yamamoto, Y., and Huang, L. (2008) Bitter peptides activate hTAS2Rs, the human bitter receptors. Biochem Biophys Res Commun 365, 851-855

40. Pronin, A. N., Tang, H., Connor, J., and Keung, W. (2004) Identification of ligands for two human bitter T2R receptors. Chem Senses 29, 583-593

41. Sainz, E., Cavenagh, M. M., Gutierrez, J., Battey, J. F., Northup, J. K., and Sullivan, S. L. (2007) Functional characterization of human bitter taste receptors. Biochem J 403, 537-543

42. Thalmann, S., Behrens, M., and Meyerhof, W. (2013) Major haplotypes of the human bitter taste receptor TAS2R41 encode functional receptors for chloramphenicol. Biochem Biophys Res Commun 435, 267-273

43. He, W., Yasumatsu, K., Varadarajan, V., Yamada, A., Lem, J., Ninomiya, Y., Margolskee, R. F., and Damak, S. (2004) Umami taste responses are mediated by alpha-transducin and alpha-gustducin. J Neurosci 24, 7674-7680

44. Prandi, S., Bromke, M., Hubner, S., Voigt, A., Boehm, U., Meyerhof, W., and Behrens, M. (2013) A subset of mouse colonic goblet cells expresses the bitter taste receptor Tas2r131. PLoS One 8, e82820

45. Wong, G. T., Gannon, K. S., and Margolskee, R. F. (1996) Transduction of bitter and sweet taste by gustducin. Nature 381, 796-800

46. Boughter, J. D., Jr., Raghow, S., Nelson, T. M., and Munger, S. D. (2005) Inbred mouse strains C57BL/6J and DBA/2J vary in sensitivity to a subset of bitter stimuli. BMC Genet 6, 36

47. Boughter, J. D., Jr., and Whitney, G. (1997) Behavioral specificity of the bitter taste gene Soa. Physiol Behav 63, 101-108

48. Capeless, C. G., Whitney, G., and Azen, E. A. (1992) Chromosome mapping of Soa, a gene influencing gustatory sensitivity to sucrose octaacetate in mice. Behav Genet 22, 655-663

49. Lush, I. E. (1984) The genetics of tasting in mice. III. Quinine. Genet Res 44, 151-160 50. Lush, I. E. (1986) The genetics of tasting in mice. IV. The acetates of raffinose, galactose and

beta-lactose. Genet Res 47, 117-123 51. Lush, I. E., and Holland, G. (1988) The genetics of tasting in mice. V. Glycine and

cycloheximide. Genet Res 52, 207-212 52. Warren, R. P., and Lewis, R. C. (1970) Taste polymorphism in mice involving a bitter sugar

derivative. Nature 227, 77-78 53. Whitney, G., and Harder, D. B. (1986) Single-locus control of sucrose octaacetate tasting among

mice. Behav Genet 16, 559-574

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


18

54. Whitney, G., and Harder, D. B. (1994) Genetics of bitter perception in mice. Physiol Behav 56, 1141-1147

55. Lee, R. J., Xiong, G., Kofonow, J. M., Chen, B., Lysenko, A., Jiang, P., Abraham, V., Doghramji, L., Adappa, N. D., Palmer, J. N., Kennedy, D. W., Beauchamp, G. K., Doulias, P. T., Ischiropoulos, H., Kreindler, J. L., Reed, D. R., and Cohen, N. A. (2012) T2R38 taste receptor polymorphisms underlie susceptibility to upper respiratory infection. J Clin Invest 122, 4145-4159

56. Hayakawa, T., Suzuki-Hashido, N., Matsui, A., and Go, Y. (2014) Frequent expansions of the bitter taste receptor gene repertoire during evolution of mammals in the Euarchontoglires clade. Mol Biol Evol 31, 2018-2031

57. Mueller, K. L., Hoon, M. A., Erlenbach, I., Chandrashekar, J., Zuker, C. S., and Ryba, N. J. (2005) The receptors and coding logic for bitter taste. Nature 434, 225-229

58. Behrens, M., Foerster, S., Staehler, F., Raguse, J. D., and Meyerhof, W. (2007) Gustatory expression pattern of the human TAS2R bitter receptor gene family reveals a heterogenous population of bitter responsive taste receptor cells. J Neurosci 27, 12630-12640

59. Caicedo, A., and Roper, S. D. (2001) Taste receptor cells that discriminate between bitter stimuli. Science 291, 1557-1560

60. Foster, S. R., Porrello, E. R., Purdue, B., Chan, H. W., Voigt, A., Frenzel, S., Hannan, R. D., Moritz, K. M., Simmons, D. G., Molenaar, P., Roura, E., Boehm, U., Meyerhof, W., and Thomas, W. G. (2013) Expression, regulation and putative nutrient-sensing function of taste GPCRs in the heart. PLoS One 8, e64579

61. Xu, J., Cao, J., Iguchi, N., Riethmacher, D., and Huang, L. (2013) Functional characterization of bitter-taste receptors expressed in mammalian testis. Mol Hum Reprod 19, 17-28

62. Nelson, T. M., Munger, S. D., and Boughter, J. D., Jr. (2005) Haplotypes at the Tas2r locus on distal chromosome 6 vary with quinine taste sensitivity in inbred mice. BMC Genet 6, 32

63. Ozeck, M., Brust, P., Xu, H., and Servant, G. (2004) Receptors for bitter, sweet and umami taste couple to inhibitory G protein signaling pathways. Eur J Pharmacol 489, 139-149

64. Hilliard, M. A., Bergamasco, C., Arbucci, S., Plasterk, R. H., and Bazzicalupo, P. (2004) Worms taste bitter: ASH neurons, QUI-1, GPA-3 and ODR-3 mediate quinine avoidance in Caenorhabditis elegans. EMBO J 23, 1101-1111

65. Oike, H., Nagai, T., Furuyama, A., Okada, S., Aihara, Y., Ishimaru, Y., Marui, T., Matsumoto, I., Misaka, T., and Abe, K. (2007) Characterization of ligands for fish taste receptors. J Neurosci 27, 5584-5592

66. Wooding, S., Bufe, B., Grassi, C., Howard, M. T., Stone, A. C., Vazquez, M., Dunn, D. M., Meyerhof, W., Weiss, R. B., and Bamshad, M. J. (2006) Independent evolution of bitter-taste sensitivity in humans and chimpanzees. Nature 440, 930-934

67. Imai, H., Suzuki, N., Ishimaru, Y., Sakurai, T., Yin, L., Pan, W., Abe, K., Misaka, T., and Hirai, H. (2012) Functional diversity of bitter taste receptor TAS2R16 in primates. Biol Lett 8, 652-656

68. Nelson, T. M., Munger, S. D., and Boughter, J. D., Jr. (2003) Taste sensitivities to PROP and PTC vary independently in mice. Chem Senses 28, 695-704

69. Harder, D. B., and Whitney, G. (1998) A common polygenic basis for quinine and PROP avoidance in mice. Chem Senses 23, 327-332

70. Biarnes, X., Marchiori, A., Giorgetti, A., Lanzara, C., Gasparini, P., Carloni, P., Born, S., Brockhoff, A., Behrens, M., and Meyerhof, W. (2010) Insights into the binding of Phenyltiocarbamide (PTC) agonist to its target human TAS2R38 bitter receptor. PLoS One 5, e12394

71. Marchiori, A., Capece, L., Giorgetti, A., Gasparini, P., Behrens, M., Carloni, P., and Meyerhof, W. (2013) Coarse-grained/molecular mechanics of the TAS2R38 bitter taste receptor: experimentally-validated detailed structural prediction of agonist binding. PLoS One 8, e64675

72. Sandau, M. M., Goodman, J. R., Thomas, A., Rucker, J. B., and Rawson, N. E. (2015) A functional comparison of the domestic cat bitter receptors Tas2r38 and Tas2r43 with their human orthologs. BMC Neurosci 16, 33

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


19

73. Lei, W., Ravoninjohary, A., Li, X., Margolskee, R. F., Reed, D. R., Beauchamp, G. K., and Jiang, P. (2015) Functional Analyses of Bitter Taste Receptors in Domestic Cats (Felis catus). PLoS One 10, e0139670

74. Matsuo, R. (2000) Role of saliva in the maintenance of taste sensitivity. Crit Rev Oral Biol Med 11, 216-229

75. Ming, D., Ruiz-Avila, L., and Margolskee, R. F. (1998) Characterization and solubilization of bitter-responsive receptors that couple to gustducin. Proc Natl Acad Sci U S A 95, 8933-8938

76. Ruiz-Avila, L., McLaughlin, S. K., Wildman, D., McKinnon, P. J., Robichon, A., Spickofsky, N., and Margolskee, R. F. (1995) Coupling of bitter receptor to phosphodiesterase through transducin in taste receptor cells. Nature 376, 80-85

77. Boughter, J. D., Harder, D. B., Capeless, C. G., and Whitney, G. (1992) Polygenic determination of quinine aversion among mice. Chemical Senses 17, 427-434

78. Bachmanov, A. A., Li, X., Li, S., Neira, M., Beauchamp, G. K., and Azen, E. A. (2001) High-resolution genetic mapping of the sucrose octaacetate taste aversion (Soa) locus on mouse Chromosome 6. Mamm Genome 12, 695-699

79. Lush, I. E., Hornigold, N., King, P., and Stoye, J. P. (1995) The genetics of tasting in mice. VII. Glycine revisited, and the chromosomal location of Sac and Soa. Genet Res 66, 167-174

80. Lush, I. E. (1982) The genetics of tasting in mice. II. Strychnine. Chemical Senses 7, 93-98 81. Shingai, T., and Beidler, L. M. (1985) Response characteristics of three taste nerves in mice.

Brain Res 335, 245-249 82. Tizzano, M., Gulbransen, B. D., Vandenbeuch, A., Clapp, T. R., Herman, J. P., Sibhatu, H. M.,

Churchill, M. E., Silver, W. L., Kinnamon, S. C., and Finger, T. E. (2010) Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals. Proc Natl Acad Sci U S A 107, 3210-3215

83. Sbarbati, A., Tizzano, M., Merigo, F., Benati, D., Nicolato, E., Boschi, F., Cecchini, M. P., Scambi, I., and Osculati, F. (2009) Acyl homoserine lactones induce early response in the airway. Anat Rec (Hoboken) 292, 439-448

84. Williams, P. (2007) Quorum sensing, communication and cross-kingdom signalling in the bacterial world. Microbiology 153, 3923-3938

85. Zhang, L. H., and Dong, Y. H. (2004) Quorum sensing and signal interference: diverse implications. Mol Microbiol 53, 1563-1571

86. Ansoleaga, B., Garcia-Esparcia, P., Llorens, F., Moreno, J., Aso, E., and Ferrer, I. (2013) Dysregulation of brain olfactory and taste receptors in AD, PSP and CJD, and AD-related model. Neuroscience 248C, 369-382

87. Garcia-Esparcia, P., Schluter, A., Carmona, M., Moreno, J., Ansoleaga, B., Torrejon-Escribano, B., Gustincich, S., Pujol, A., and Ferrer, I. (2013) Functional genomics reveals dysregulation of cortical olfactory receptors in Parkinson disease: novel putative chemoreceptors in the human brain. J Neuropathol Exp Neurol 72, 524-539

88. Dehkordi, O., Rose, J. E., Fatemi, M., Allard, J. S., Balan, K. V., Young, J. K., Fatima, S., Millis, R. M., and Jayam-Trouth, A. (2012) Neuronal expression of bitter taste receptors and downstream signaling molecules in the rat brainstem. Brain Res 1475, 1-10

89. Singh, N., Vrontakis, M., Parkinson, F., and Chelikani, P. (2011) Functional bitter taste receptors are expressed in brain cells. Biochem Biophys Res Commun 406, 146-151

90. Li, F., and Zhou, M. (2012) Depletion of bitter taste transduction leads to massive spermatid loss in transgenic mice. Mol Hum Reprod 18, 289-297

91. Voigt, A., Hubner, S., Lossow, K., Hermans-Borgmeyer, I., Boehm, U., and Meyerhof, W. (2012) Genetic labeling of Tas1r1 and Tas2r131 taste receptor cells in mice. Chem Senses 37, 897-911

92. Clark, A. A., Dotson, C. D., Elson, A. E., Voigt, A., Boehm, U., Meyerhof, W., Steinle, N. I., and Munger, S. D. (2015) TAS2R bitter taste receptors regulate thyroid function. FASEB J 29, 164-172

93. Deckmann, K., Filipski, K., Krasteva-Christ, G., Fronius, M., Althaus, M., Rafiq, A., Papadakis, T., Renno, L., Jurastow, I., Wessels, L., Wolff, M., Schutz, B., Weihe, E., Chubanov, V.,

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


20

Gudermann, T., Klein, J., Bschleipfer, T., and Kummer, W. (2014) Bitter triggers acetylcholine release from polymodal urethral chemosensory cells and bladder reflexes. Proc Natl Acad Sci U S A 111, 8287-8292

94. Correia, J. N., Conner, S. J., and Kirkman-Brown, J. C. (2007) Non-genomic steroid actions in human spermatozoa. "Persistent tickling from a laden environment". Semin Reprod Med 25, 208-219

95. Boughter, J. D., Jr., St John, S. J., Noel, D. T., Ndubuizu, O., and Smith, D. V. (2002) A brief-access test for bitter taste in mice. Chem Senses 27, 133-142

96. Dotson, C. D., and Spector, A. C. (2004) The relative affective potency of glycine, L-serine and sucrose as assessed by a brief-access taste test in inbred strains of mice. Chem Senses 29, 489-498

97. Glendinning, J. I., Gresack, J., and Spector, A. C. (2002) A high-throughput screening procedure for identifying mice with aberrant taste and oromotor function. Chem Senses 27, 461-474

98. Zhang, Y., Hoon, M. A., Chandrashekar, J., Mueller, K. L., Cook, B., Wu, D., Zuker, C. S., and Ryba, N. J. (2003) Coding of sweet, bitter, and umami tastes: different receptor cells sharing similar signaling pathways. Cell 112, 293-301

99. Livak, K. J., and Schmittgen, T. D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408

100. Barratt-Fornell, A., and Drewnowski, A. (2002) The Taste of Health: Nature's Bitter Gifts. Nutr Today 37, 144-150

101. Belitz, H. D., and Wieser, H. (1985) Bitter compounds: Occurrence and structure‐activity relationships. Food Reviews International 1, 271-354

102. Drewnowski, A., and Gomez-Carneros, C. (2000) Bitter taste, phytonutrients, and the consumer: a review. Am J Clin Nutr 72, 1424-1435

103. Maga, J. A. (1990) Compound structure versus bitter taste. In: Rousseff R. L. (ed.) Bitterness in foods and beverages; developments in food science 25. Elsevier. Amsterdam, 35–48.

104. Rouseff, R. L. (1990) Bitterness in food products - an overview. In: Rousseff R. L. (ed.) Bitterness in foods and beverages; developments in food science 25. Elsevier. Amsterdam, 1-14

105. Wiener, A., Shudler, M., Levit, A., and Niv, M. Y. (2012) BitterDB: a database of bitter compounds. Nucleic Acids Res 40, D413-419

106. Tizzano, M., Cristofoletti, M., Sbarbati, A., and Finger, T. E. (2011) Expression of taste receptors in solitary chemosensory cells of rodent airways. BMC Pulm Med 11, 3

107. Appendino, G., Belliardo, F., Nano, G. M., and Stefenelli, S. (1982) Sesquiterpene Lactones from Artemisia-Genipi Weber - Isolation and Determination in Plant-Material and in Liqueurs. Journal of Agricultural and Food Chemistry 30, 518-521

108. Appendino, G., Gariboldi, P., and Menichini, F. (1991) The stereochemistry of arglabin, a cytotoxic guaianolide from Artemisia myriantha. Fitoterapia 62, 275-276

109. Appendino, G., Nano, G. M., Calleri, M., and Chiari, G. (1986) The Crystal-Structures of Spiciformin Acetate and Tatridin-B Dibenzoate. Gazzetta Chimica Italiana 116, 57-61

110. Fattorusso, E., Taglialatela-Scafati, O., Campagnuolo, C., Santelia, F. U., Appendino, G., and Spagliardi, P. (2002) Diterpenoids from Cascarilla (Croton eluteria Bennet). J Agric Food Chem 50, 5131-5138

111. Wyrembek, P., Negri, R., Kaczor, P., Czyzewska, M., Appendino, G., and Mozrzymas, J. W. (2012) Falcarindiol allosterically modulates GABAergic currents in cultured rat hippocampal neurons. J Nat Prod 75, 610-616

112. Beauhaire, J., Fourrey, J. L., Vuilhorgne, M., and Lallemand, J. Y. (1980) Dimeric Sesquiterpene Lactones - Structure of Absinthin. Tetrahedron Letters 21, 3191-3194

113. Kohl, S., Behrens, M., Dunkel, A., Hofmann, T., and Meyerhof, W. (2013) Amino acids and peptides activate at least five members of the human bitter taste receptor family. J Agric Food Chem 61, 53-60

114. Ammon, C., Schafer, J., Kreuzer, O. J., and Meyerhof, W. (2002) Presence of a plasma membrane targeting sequence in the amino-terminal region of the rat somatostatin receptor 3. Arch Physiol Biochem 110, 137-145

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


21

115. Slack, J. P., Brockhoff, A., Batram, C., Menzel, S., Sonnabend, C., Born, S., Galindo, M. M., Kohl, S., Thalmann, S., Ostopovici-Halip, L., Simons, C. T., Ungureanu, I., Duineveld, K., Bologa, C. G., Behrens, M., Furrer, S., Oprea, T. I., and Meyerhof, W. (2010) Modulation of bitter taste perception by a small molecule hTAS2R antagonist. Curr Biol 20, 1104-1109

116. Ueda, T., Ugawa, S., Yamamura, H., Imaizumi, Y., and Shimada, S. (2003) Functional interaction between T2R taste receptors and G-protein alpha subunits expressed in taste receptor cells. J Neurosci 23, 7376-7380

117. Krautwurst, D., Yau, K. W., and Reed, R. R. (1998) Identification of ligands for olfactory receptors by functional expression of a receptor library. Cell 95, 917-926

118. Yu, Y., de March, C. A., Ni, M. J., Adipietro, K. A., Golebiowski, J., Matsunami, H., and Ma, M. (2015) Responsiveness of G protein-coupled odorant receptors is partially attributed to the activation mechanism. Proc Natl Acad Sci U S A 112, 14966-14971

119. Mouse Genome Sequencing, C., Waterston, R. H., Lindblad-Toh, K., Birney, E., Rogers, J., Abril, J. F., Agarwal, P., Agarwala, R., Ainscough, R., Alexandersson, M., An, P., Antonarakis, S. E., Attwood, J., Baertsch, R., Bailey, J., Barlow, K., Beck, S., Berry, E., Birren, B., Bloom, T., Bork, P., Botcherby, M., Bray, N., Brent, M. R., Brown, D. G., Brown, S. D., Bult, C., Burton, J., Butler, J., Campbell, R. D., Carninci, P., Cawley, S., Chiaromonte, F., Chinwalla, A. T., Church, D. M., Clamp, M., Clee, C., Collins, F. S., Cook, L. L., Copley, R. R., Coulson, A., Couronne, O., Cuff, J., Curwen, V., Cutts, T., Daly, M., David, R., Davies, J., Delehaunty, K. D., Deri, J., Dermitzakis, E. T., Dewey, C., Dickens, N. J., Diekhans, M., Dodge, S., Dubchak, I., Dunn, D. M., Eddy, S. R., Elnitski, L., Emes, R. D., Eswara, P., Eyras, E., Felsenfeld, A., Fewell, G. A., Flicek, P., Foley, K., Frankel, W. N., Fulton, L. A., Fulton, R. S., Furey, T. S., Gage, D., Gibbs, R. A., Glusman, G., Gnerre, S., Goldman, N., Goodstadt, L., Grafham, D., Graves, T. A., Green, E. D., Gregory, S., Guigo, R., Guyer, M., Hardison, R. C., Haussler, D., Hayashizaki, Y., Hillier, L. W., Hinrichs, A., Hlavina, W., Holzer, T., Hsu, F., Hua, A., Hubbard, T., Hunt, A., Jackson, I., Jaffe, D. B., Johnson, L. S., Jones, M., Jones, T. A., Joy, A., Kamal, M., Karlsson, E. K., Karolchik, D., Kasprzyk, A., Kawai, J., Keibler, E., Kells, C., Kent, W. J., Kirby, A., Kolbe, D. L., Korf, I., Kucherlapati, R. S., Kulbokas, E. J., Kulp, D., Landers, T., Leger, J. P., Leonard, S., Letunic, I., Levine, R., Li, J., Li, M., Lloyd, C., Lucas, S., Ma, B., Maglott, D. R., Mardis, E. R., Matthews, L., Mauceli, E., Mayer, J. H., McCarthy, M., McCombie, W. R., McLaren, S., McLay, K., McPherson, J. D., Meldrim, J., Meredith, B., Mesirov, J. P., Miller, W., Miner, T. L., Mongin, E., Montgomery, K. T., Morgan, M., Mott, R., Mullikin, J. C., Muzny, D. M., Nash, W. E., Nelson, J. O., Nhan, M. N., Nicol, R., Ning, Z., Nusbaum, C., O'Connor, M. J., Okazaki, Y., Oliver, K., Overton-Larty, E., Pachter, L., Parra, G., Pepin, K. H., Peterson, J., Pevzner, P., Plumb, R., Pohl, C. S., Poliakov, A., Ponce, T. C., Ponting, C. P., Potter, S., Quail, M., Reymond, A., Roe, B. A., Roskin, K. M., Rubin, E. M., Rust, A. G., Santos, R., Sapojnikov, V., Schultz, B., Schultz, J., Schwartz, M. S., Schwartz, S., Scott, C., Seaman, S., Searle, S., Sharpe, T., Sheridan, A., Shownkeen, R., Sims, S., Singer, J. B., Slater, G., Smit, A., Smith, D. R., Spencer, B., Stabenau, A., Stange-Thomann, N., Sugnet, C., Suyama, M., Tesler, G., Thompson, J., Torrents, D., Trevaskis, E., Tromp, J., Ucla, C., Ureta-Vidal, A., Vinson, J. P., Von Niederhausern, A. C., Wade, C. M., Wall, M., Weber, R. J., Weiss, R. B., Wendl, M. C., West, A. P., Wetterstrand, K., Wheeler, R., Whelan, S., Wierzbowski, J., Willey, D., Williams, S., Wilson, R. K., Winter, E., Worley, K. C., Wyman, D., Yang, S., Yang, S. P., Zdobnov, E. M., Zody, M. C., and Lander, E. S. (2002) Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520-562

120. Levy, S., Sutton, G., Ng, P. C., Feuk, L., Halpern, A. L., Walenz, B. P., Axelrod, N., Huang, J., Kirkness, E. F., Denisov, G., Lin, Y., MacDonald, J. R., Pang, A. W. C., Shago, M., Stockwell, T. B., Tsiamouri, A., Bafna, V., Bansal, V., Kravitz, S. A., Busam, D. A., Beeson, K. Y., McIntosh, T. C., Remington, K. A., Abril, J. F., Gill, J., Borman, J., Rogers, Y.-H., Frazier, M. E., Scherer, S. W., Strausberg, R. L., and Venter, J. C. (2007) The Diploid Genome Sequence of an Individual Human. PLoS Biol 5, e254

121. Feng, D.-F., and Doolittle, R. (1987) Progressive sequence alignment as a prerequisitetto correct phylogenetic trees. Journal of Molecular Evolution 25, 351-360 LA - English

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


22

122. Jeanmougin, F., Thompson, J. D., Gouy, M., Higgins, D. G., and Gibson, T. J. (1998) Multiple sequence alignment with Clustal X. Trends in Biochemical Sciences 23, 403-405

123. Huerta-Cepas, J., Dopazo, J., and Gabaldon, T. (2010) ETE: a python Environment for Tree Exploration. BMC Bioinformatics 11, 24

124. Ballesteros, J. A., and Weinstein, H. (1995) Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. Methods Neuroscience 25, 366–428

Footnotes Funding This study has been financed by the Federal Ministry for Education and Research, the Ministry for Science, Research and Culture of the state of Brandenburg, Germany as well as the European Union’s Seventh Framework Programme for research, technological development and demonstration (#613879; SynSignal). Abbrevations The abbreviations used are: AHL, acyl-homoserine lactone; ECL, extracellular loops; FLIPR, fluorescence image plate reader; GPCR, G protein-coupled receptors; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HSL, homoserine lactone; HSV, herpes simplex virus glycoprotein D epitope; ICL, intracellular loops; PEITC, phenyl ethyl isothiocyanate; PROP, 6-propyl-2-thiouracil; PTC, phenylthiocarbamide; qRT-PCR, quantitative RT-PCR; Qui, quinine locus; RUA, raffinose undecaacetate; RT-PCR, reverse transcribed polymerase chain reaction; SCC, solitary chemosensory cells; SOA, sucrose octaacetate; sst3, somatostatin receptor subtype 3; TAS2Rs, human bitter taste receptor class 2; Tas2rs, murine bitter taste receptor class 2; TM, transmembrane domains Figure legends Figure 1. Tas2rs are expressed in the posterior lingual papillae. (A) Expression levels of all 35 Tas2rs and α-gustducin in epithelium enriched in vallate and foliate papillae of C57BL/6 mice determined by quantitative RT-PCR (means ± SE, n = 4, normalized to β-actin). (B) In situ hybridization using digoxigenin-labeled cRNA probes specific for selected Tas2rs and gustducin (Gα-Gust) revealed the presence of corresponding RNAs in vallate papillae of C57BL/6 mice (scale bar, 50 µm). Figure 2. Calcium responses of Tas2r-expressing cells challenged with different bitter compounds. (A) For calcium imaging experiments, cDNA constructs coding for the indicated Tas2rs or empty vector (pcDNA5 or pEAK10 = MOCK) were expressed in HEK293T-Gα16gust44 cells, seeded in 96-well microtiter plates. Calcium traces were recorded in an automated fluorometric imaging plate reader (FLIPRtetra) based on the detection of changes in fluorescence after the application of the indicated bitter compounds (first stimulus). A second application of 100 nM somatostatin-14 activating endogenous somatostatin receptors served as vitality control. Arrows point to cellular responses specific to bitter compound stimulation. Specificity of receptor activation was controlled by application of same substances on MOCK-transfected cells. Furthermore, the reliability of the experiment was checked every time by running parallel assays for human TAS2Rs with known activation patterns (right columns). (B) Exemplary magnified calcium traces for indicated bitter receptors and MOCK after application of denatonium saccharide (first stimulus). To demonstrate cell vitality a second application with a final concentration of 100 nM somatostatin-14 was included to activate endogenous somatostatin receptors. Figure 3. Concentration-response relations of Tas2r105-expressing cells stimulated with increasing concentrations of the indicated compounds calculated from calcium traces acquired by FLIPR recordings. Changes in fluorescence (ΔF/F) were plotted semi-logarithmically versus agonist concentrations. Figure 4. Concentration-response relations for denatonium benzoate in cells expressing Tas2r105, Tas2r123, Tas2r135, Tas2r140, or Tas2r144. The curves have been deduced from calcium traces

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


23

monitored in FLIPR experiments. Changes in fluorescence (ΔF/F) were plotted semi-logarithmically versus agonist concentrations. Figure 5. Intracellular calcium traces of Tas2r105 (non-taster variant) in HEK293T cells stable expressing Gα15 (continuous line) and Gα16gust44 (dashed line) recorded in FLIPR experiments upon stimulation with six exemplary compounds, indicating that low efficacy activators of Tas2r105 resulted in lower or even absent responses in Gα15-expressing cells. Calibration bar denotes 2000 relative light units (y) and 1 min (x). Figure 6. Confocal fluorescence images of HEK293T Gα16gust44 cells transiently transfected with cDNAs of mouse bitter taste receptors. Cells were transfected with Rho-tagged Tas2r constructs and underwent immunostaining with Rho-antibody (green) either after (left panel) or before fixation (right panel). The cell surface is visualized by biotin-conjugated concanavalin A and streptavidin conjugated Alexa fluor 633 (red). Taste receptors located on the cell surface appear yellow in the overlay (merge) and are exemplarily indicated by orange arrows. Receptors without surface expression are exemplarily indicated by white arrows. Figure 7. Phylogenetic tree based on alignment of amino acid sequences for mouse and human Tas2rs and their chromosomal localization. After a multiple sequence alignment of the Tas2rs manually adjusted to consider the potential tertiary structure, a neighbor-joining tree was constructed. Percentage bootstrap values higher than 50 are displayed as node labels. Tas2rs are classified into 5 clusters. Muroid cluster I represents a species- and lineage-specific cluster of 11 mouse receptors and human TAS2R14, whereas muroid cluster I and II comprise 5 mouse Tas2rs connected to TAS2R10 and 3 mouse Tas2rs associated with TAS2R13, respectively. The anthropoid cluster represents human cluster composed of 8 TAS2RS related to the glires cluster II comprised of Tas2r120 and Tas2r136. Figure 8. Comparisons of ligand-receptor interactions for orthologous human and mouse Tas2rs for bitter compounds that activate at least one receptor. (A) Ligand-receptor responses of one-to-one orthologous TAS2Rs. (B) Agonist spectra for species-specific Tas2r clusters and associated Tas2rs in the other species. Mice show three such species- or lineage-specific expansions associated with human TAS2R14 (Glires cluster I), TAS2R10 (muroid cluster I), or TAS2R13 (muroid cluster II), whereas humans have only one cluster associated with mouse Tas2r120 and Tas2r136 (anthropoid cluster). Figure 9. Taxonomic tree of selected Euarchontoglires and Carnivora species (upper left panel). The tree was generated using the Common Tree Software of the Taxonomy Browser of the NCBI website (www.ncbi.nlm.nih.gov) with Tree Viewer software (123). Yellow lines indicate hypothesized evolutionary origin of TAS2R38 orthologs with low sensitive PTC recognition and lack of PROP responsiveness. Green lines indicate assumed evolutionary origin of high sensitive PTC and PROP detecting TAS2R38 orthologs, whereas red lines label the origin of PTC/PROP insensitive TAS2R38 orthologs found in rats and mice. Phylogenetic tree of functionally characterized TAS2R38 orthologs (upper right panel). The low PTC sensitive cat Tas2r38 cDNA (yellow) (72), the two high PTC and PROP sensitive human (33) and chimpanzee (66) TAS2R38 cDNAs as well as the PTC/PROP insensitive Tas2r138 cDNAs of mouse (this study) were aligned with AlignX of the Vector NTI software. Comparison of functionally critical residues in selected TAS2R38 orthologs (lower panel). A subset of species shown in A) was analyzed for functionally critical amino acid positions in the corresponding TAS2R38 orthologs. The first three rows refer to amino acid positions found in human PTC/PROP taster and non-taster variants of this receptor. The first position is located in the first intracellular loop (IC Position 49). The second and third rows specify residues in the 6th and 7th transmembrane domain, respectively, by their position according to the Ballesteros-Weinstein nomenclature (124) followed by functionally important residues in the binding pocket of human TAS2R38 contributing to PROP and PTC activation (70,71). Among these residues were tryptophane 201, serine 260, and phenylalanine 264, the corresponding positions are depicted in rows 4 (5.46), 5 (6.52), and 6 (6.56).

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


24

Figure 10. Amino acid sequence alignment of murine cluster I receptors (upper panel). The alignment was created using the Align X program of the Vector NTI software (Life Technologies). Residues are labeled according to the degree of conservation (yellow, identical; green, conserved; blue identical in at least half of the sequences). Transmembrane domains (TM) are labeled by red boxes and numbered. The orientation in the lipid bilayer is shown by arrows (arrowheads point towards the extracellular site). Intra- (ICL) and extracellular loops (ECL) are indicated and numbered. Amino acid positions demonstrated to reside in the ligand binding pocket of human TAS2R10 are indicated by stars. Red stars highlight amino acid positions with pronounced agonist selectivity, black stars contribute to ligand binding and binding pocket constitution (30). Comparison of functionally important receptor positions (lower panel). The six receptors belonging to murine cluster I were compared for their conservation at positions experimentally proven to contribute to agonist binding in the human TAS2R10, a member of this cluster. Receptor residues are indicated by their position according to the Ballesteros-Weinstein numbering system (124). Red labeled residues reside in positions exhibiting pronounced agonist selectivity in TAS2R10, other positions were demonstrated to constitute the receptor binding site (30). Figure 11. Selected concentration-response functions of cells transiently transfected with DNA for orthologous mouse and human Tas2rs. Functions are based on calcium traces acquired by FLIPR recordings. Changes in fluorescence (ΔF/F) were plotted semi-logarithmically versus agonist concentrations. Tables Table 1. Threshold concentrations [mM] for Tas2r activators. Specific compounds for mouse Tas2rs are shown in bold. Substances activating only single receptors are underscored. -, no response.

105

108

109

110

113

114

115

117

119

120

121

122

123

125

126

135

137

138

139

140

144

2-Heptyl-3-hydroxy-quinolone 0.003 - - - - - - - - - 0.003 - - - - - - - - - -5-Propyl-2-thiouracil - - - - - - - - - - 0.1 - - - - 0.1 - 0.1 - - - 6-Propyl-2-thiouracil 0.3 1 - - - - - - - 0.3 0.3 - - - - 0.3 1 - - - - Absinthin - - - 0.03 - - - - - - - - 0.1 - - - - - - 0.1 -Acesulfame K - - - - - - - - - - - - - - - 0.3 - - - - -Acetylpyrazine - - - - - - - - - - 1 - - - - - - - - - - Allylisothiocyanate - - - - - - - - - - - - - - - 0.3 - - - - - Amarogentin 0.0003 1 - - - - - - - - - - 0.3 - - - - - - - 1Arborescin - - - 0.03 - - - - 0.01 - 0.01 - - - - - - - - - - Arbutin - - - - - - - - - - - - - - 30 - - - - - - Arglabin 0.1 - - 0.03 - - - - 0.1 - - - - - - - - - - - - Artemorin 0.03 0.1 0.1 - - - - - - - - - - - - - - - - - -Azathioprine - - - - - - - - - - 0.3 - - - - - - - - - - Brucine - - - - - - - 0.001 - - - - - - - - - - - - - Caffeine - - - - - - - - - - 0.01 - - - - - - - - - - Camphor - - - - - - - - 1 - - - - - - - 1 - - - 1Carisoprodol 0.003 - - - - - - - - - - - - - - - - - - - -Carotene, β- 0.001 - - - - - - - - - - - - - - - - - - - - Chloramphenicol 0.03 - - - - - - - 0.3 - - - - - - - - - - - - Chloroquine - - - - - - 0.1 - - - - - - - - - - - - - - Chlorpheniramine - 0.1 - - - - - - - - - - - - - - - - - 0.03 0.1 Cnicin - - - - - - - 0.1 - - - - - - - - - - - - - Colchicine - - 3 - - - - - - - - - - - 3 - - - - - 3 Costunolide 0.01 - - - - - - - 0.03 0.03 - - - - - - - - - - - Coumarin 0.3 - - - - - - - - - - - - - - - - - - - - Cucurbitacin B 0.03 - - - - 0.003 - - - - - - - - - - - - - - - Cucurbitacin D 0.1 - - - - 0.01 - - - - - - - - - - -Cucurbitacin E 0.01 - - - - 0.003 - - - - - - - - - - - - - - -Cucurbitacin I 0.15 - - - - 0.001 - - - - - - - - - - - - - - - Cycloheximide 0.00001 - - - - - - - - - - - - - - - - - - - - Denatonium benzoate 0.1 - - - - - - - - - - - 0.3 - - 0.1 - - - 0.3 3Denatonium saccharide 0.1 - - - - - - - - - - - 0.3 - - 0.1 - - - 0.3 3Diphenidol 0.01 0.1 - 0.1 - - - - - - - - 0.01 - - - 0.1 - - - 0.1 Emetine - 0.03 - - - - - - - - - - - - - - - - - 0.03 - Epicatechin - - - - - - - - - - - - - - 1 - - - - - 1 Epigallocatechin gallate - - - - - - - - - - - - - - - - - - - - 0.01Erythromycin - - - - - - - - - - - - 0.3 - - - - - - - - Ethylpyrazine - - - - - - - - - - 3 - - - - - - - - - 3 Falcarindiol 0.01 - - - - - - - - - - - - - - - - - - - - Famotidine - - - - - - - - - - 0.03 - - - - - - - - - -Haloperidol - - - 0.001 - - - - - - - - - - - - - - - - 0.03Helicin - - - - - - - - - - - - - - 10 - - - - - - HSL, 3-oxo-C6- 0.1 - - - - - - - - - - - - - - - - - - - -HSL, 3-oxo-C8- 0.1 - - - - - - - - - - - - - - - - - - - -

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


25

105

108

109

110

113

114

115

117

119

120

121

122

123

125

126

135

137

138

139

140

144

HSL, C4- 1 - - - - - - - - - - - - - - - - - - - - HSL, C6- 0.3 - - - - - - - - - - - - - - - - - - - - Humulone 0.001 - - - - - - - - - - - - - - - - - - - -Lidocaine 0.01 - - - - - - - - - - - - - - - - - - - - Orphenadrine 0.03 - - - - - - - - - - - - - - - - - - 0.003 0.03Ouabain - - - - - - - - - - - - - - - - - - - - 3Papaverine - - - - - - - - - - - - - - - - - - - - 0.01Parthenolide 0.1 - - - - - - - - - - - - - - - - - - - - Phenanthroline 1 - - - - - - - - - 1 1 - - - 1 - - - - 1 Phenyl-β-D-glucopyranoside - - - - - - - - - - - - - 3 10 - - - - - -Phenylbutazone 0.1 - - - 0.001 - - - - - - - - - - - - - - - - Phenylethyl isothiocyanate 0.03 - - - - - - - - - - - - - - - - - - - - Picrotin 3 - - - - 1 - - - - - - - - - - - - - - - Picrotoxinin 0.3 - - - - 3 - - - - - - - - - - - - - - -Progesterone - - - 0.001 - 0.003 - - - - - - - - - - - - - - - Pyrocatechin 0.3 - - - - - - - - - - - - - - - - - - - 1 Quassin 0.3 - - - - - - - - - - - - - - - - - - - - Quinine 0.01 0.01 - - - - 0.003 - - - - - - - 0.01 - 0.01 - - 0.003 0.01RUA - - 0.3 0.3 - - - - - - - - 0.3 - - - - - - - - Saccharin 1 - 3 - - - - - - - - - - - - 0.1 - - - - 10 Salicin, D- - - - - - - - - - - - - - - 10 - - - - - -Salicylic acid - - - - - - - - - - - - - - - 0.01 - - - - -Santonin 0.3 - - - - 0.3 - - - - - - - - - - - - - - - Sinigrin 1 - - - - - - - - - - - - - - - - - - - - SOA - - - - - - - 0.03 - - - - - - - - - - - - -Sodium benzoate 3 - - - - - - - - - 10 - - - - 0.03 - - - - - Strychnine - - - - - - - 0.01 - - - - - - - - - - - 0.01 - Sucralose 10 - 30 - - - 3 3 - - - - 30 - - - - - 30 - 10 Tatridin A 0.3 - - - - - - - - - - - - - - - - - - - -Taurocholic acid - - - - - - - 0.03 - - - - 0.3 - - - - - - - 0.3 Theobromine - - - - - - - - - - 0.03 - - - - - - - - - - Theophylline - - - - - - - - - - 0.03 - - - - - - - - - - Thujone, α- 0.03 - - - - - - - 0.03 - - - - - - - - - - - -Umbelliferone 0.6 - - - - - - - - - - - - 0.6 - - - - - - -Xanthotoxin 0.01 - - - - - - - 0.1 0.03 0.1 - - - - 0.1 - - - - - Yohimbine 0.3 0.3 - - - - - - - - - - 0.1 - - - - 0.3 - - 0.3

Table 2. Lick ratios (mean ± SE; n = 8) of C57BL/6 mice to concentration series of 22 bitter compounds. Statistical significance was determined by analysis of mixed models and post-hoc-analysis. Thereby, identical letters indicate no variances, whereas variable letters represent significant differences to at least a significance level < 0.05.

substance concentration [mM] substance/water lick ratio

Acesulfame K

1 0.94 ± 0.06a

10 0.80 ± 0.13a

100 0.85 ± 0.06a

1000 0.05 ± 0.01b

Aloin

0.001 0.95 ± 0.08a

0.01 1.01 ± 0.08a

0.1 0.71 ± 0.13b

1 0.41 ± 0.14b,c

Amygdalin

0.03 0.86 ± 0.07a

0.3 0.78 ± 0.14a,b

3 0.62 ± 0.15a,b,c

30 0.51 ± 0.15b,c

100 0.33 ± 0.15c

Atropine

0.01 0.95 ± 0.07a

0.1 0.85 ± 0.12a

1 0.31 ± 0.12b

10 0.06 ± 0.02c

Berberine

0.001 0.81 ± 0.13a

0.01 0.84 ± 0.15a

0.1 0.92 ± 0.09a

1 0.10 ± 0.03b

Brucine 0.01 0.87 ± 0.12a

0.1 0.84 ± 0.12a

1 0.56 ± 0.16b

Caffeine

0.1 0.76 ± 0.13a

1 0.91 ± 0.11a

10 0.80 ± 0.14a

100 0.19 ± 0.05b

Chloroquine

0.1 0.64 ± 0.09a

1 0.25 ± 0.07b

10 0.06 ± 0.02c

100 0.04 ± 0.01c

Colchicine 0.0003 0.91 ± 0.10a

0.003 0.79 ± 0.10a

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


26

substance concentration [mM] substance/water lick ratio 0.03 0.32 ± 0.10b

0.3 0.07 ± 0.02c

3 0.08 ± 0.02c

30 0.04 ± 0.01c

Denatonium benzoate

0.03 0.75 ± 0.14a

0.3 0.82 ± 0.14a

3 0.06 ± 0.02b

30 0.05 ± 0.01b

Naringin

0.001 0.93 ± 0.11a

0.01 0.80 ± 0.14a,b

0.1 0.64 ± 0.14b

1 0.52 ± 0.15b

Nicotine

0.01 0.94 ± 0.10a

0.1 0.86 ± 0.14a

1 0.76 ± 0.13a

10 0.29 ± 0.08b

Ouabain 0.3 0.69 ± 0.16a

3 0.28 ± 0.13b

30 0.12 ± 0.07b

Phenanthroline 0.1 0.82 ± 0.13a

1 0.47 ± 0.13b

10 0.12 ± 0.04c

Phenyl-β-D-glucopyranoside

0.1 0.78 ± 0.11a

1 0.67 ± 0.14a

10 0.35 ± 0.15b

100 0.07 ± 0.01c

PEITC

0.003 0.78 ± 0.12a

0.03 0.94 ± 0.11a

0.3 0.69 ± 0.18a

3 0.66 ± 0.15a

PTC

0.01 1.01 ± 0.06a

0.1 0.95 ± 0.09a

1 0.67 ± 0.15b

10 0.30 ± 0.13c

Quinine

0.001 0,98 ± 0.08a

0.01 0.87 ± 0.07a

0.1 0.23 ± 0.08b

1 0.08 ± 0.02b

D-(-)-Salicin 1 1.04 ± 0.09a

10 0.69 ± 0.17b

100 0.12 ± 0.07c

SOA 0.03 0.84 ± 0.12a,c

0.3 0.71 ± 0.18b,c

3 0.38 ± 0.31b

Thiamine

0.1 0.83 ± 0.11a

1 0.96 ± 0.13a

10 0.26 ± 0.08b

100 0.04 ± 0.01c

Thujone 0.03 0.96 ± 0.11a

0.3 0.90 ± 0.14a

3 0.47 ± 0.14b

Table 3. Cell surface localization experiments with HEK293T Gα16gust44 cells transiently transfected with Rho-tagged cDNAs of mouse bitter taste receptors before and after permeabilization. +, expressed; -, no expression detectable.

Receptor Before permeabilization After permeabilization Tas2r102 - + Tas2r105 + + Tas2r106 + + Tas2r108 + + Tas2r114 + + Tas2r118 + + Tas2r119 + + Tas2r120 + + Tas2r121 + + Tas2r123 + + Tas2r126 + + Tas2r129 + + Tas2r131 - + Tas2r134 + + Tas2r144 + + MOCK - -

Figures

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Maik Behrens and Wolfgang MeyerhofKristina Lossow, Sandra Hübner, Natacha Roudnitzky, Jay P. Slack, Federica Pollastro,

receptive ranges for orthologous receptors in mice and humansComprehensive analysis of mouse bitter taste receptors reveals different molecular

published online May 20, 2016J. Biol. Chem.

10.1074/jbc.M116.718544Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

http://www.jbc.org/content/suppl/2016/05/20/M116.718544.DC1

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M116.718544

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M116.718544v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2016/05/20/jbc.M116.718544

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2016/05/20/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2016/05/20/jbc.M116.718544

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/suppl/2016/05/20/M116.718544.DC1

http://www.jbc.org/

Documents

Deorphanization of mouse bitter taste receptors · 5/20/2016 · bitter taste receptor genes. It is assumed that the orthologous bitter taste receptor genes mediate the recognition